The combination of time-resolved and spectral resolved techniques as achieved by SLIM (spectrally resolved fluorescence lifetime imaging) improves the analysis of complex situations, when different fluorophores have to be distinguished. This could be the case when endogenous fluorophores of living cells and tissues are observed to identify the redox state and oxidative metabolic changes of the mitochondria. Other examples are FRET (resonant energy transfer) measurements, when different donor/acceptor pairs are observed simultaneously. SLIM is working in the time domain employing excitation with short light pulses and detection of the fluorescence intensity decay in many cases with time-correlated single photon counting (TCSPC). Spectral resolved detection is achieved by a polychromator in the detection path and a 16-channel multianode photomultiplier tube with the appropriate routing electronics. Within this paper special attention will be focused on FRET measurements with respect to protein interactions in Alzheimers disease. Using global analysis as the phasor plot approach or integration of the kinetic equations taking into account the multidimensional datasets in every spectral channel we could demonstrate considerable improvement of our calculations.

In many fields of life science, visualization of spatial proximity, as an indicator of protein interactions in living cells, is of outstanding interest. A method to accomplish this is the measurement of Förster resonant energy transfer (FRET) by means of spectrally resolved fluorescence lifetime imaging microscopy. The fluorescence lifetime is calculated using a multiple-wavelength fitting routine. The donor profile is assumed first to have a monoexponential time-dependent behavior, and the acceptor decay profile is solved analytically. Later, the donor profile is assumed to have a two-exponential time-dependent behavior and the acceptor decay profile is derived analytically. We develop and apply a multispectral fluorescence lifetime imaging microscopy analysis system for FRET global analysis with time-resolved and spectrally resolved techniques, including information from donor and acceptor channels in contrast to using just a limited spectral data set from one detector only and a model accounting only for the donor signal. This analysis is used to demonstrate close vicinity of β-secretase (BACE) and GGA1, two proteins involved in Alzheimer's disease pathology. We attempt to verify if an improvement in calculating the donor lifetimes could be achieved when time-resolved and spectrally resolved techniques are simultaneously incorporated.

Essential for the proteolytic processing of APP by BACE1 - and therewith for the generation of Abeta - is the transport and sorting of both proteins through endosomal and Golgi compartments. The family of Golgi-localized γ-ear-containing ARF-binding (GGA) proteins was shown to have striking functions in cargo sorting in these pathways. Recently, it was shown that GGA1 and GGA3 can interact with BACE1, that they are expressed in neurons and GGA3 is reduced in AD brain tissues. The GGA VHS-domain was found to be the major binding motif necessary for interaction with BACE1. We applied Immunoprecipitation and Fluorescence Lifetime imaging Microscopy (FLIM) with different GGA-domain deletions to analyze VHS-domain independent BACE1-GGA interaction and the binding capacity of the different deletion mutants. We further extended our approach by ELISA assays and Western Blot analysis to measure the influence on APP processing. Co-immunoprecipitation experiments showed that all GGA-VHS deletion-mutants can still be precipitated by BACE1, though to a lower extent than their wild type forms. Additionally, in FLIM experiments the lifetime of GGA-VHS deletion-mutants was only slightly increased compared to wild-type GGAs. Furthermore, cells co-transfected with APP, BACE and either wild-type GGAs or VHS deletion-mutants showed no significant differences in APP processing in Mesoscale Elisa assays and Western Blot analysis. Therefore, we extended our approach to identify other domains responsible for GGA-BACE1 interaction using several GGA domain-deletions and point-mutations to identify the major binding motif necessary for GGA-BACE interaction. In conclusion, our data suggest that the GGA VHS-domain has only limited influence upon the processing of APP and upon interaction with and trafficking of BACE1. As all other known GGA cargo proteins seem to be dependent on the VHS-domain differing BACE1 interactions are of high interest in the attempt to find BACE1 specific transport and trafficking modifiers.

Parkinson's disease (PD) is a progressive-neurodegenerative disorder that affects more than 6 million people around the world. However, conventional techniques for PD detection are often hand-crafted, in which special expertise is needed. In this study, considering the importance of rapid diagnosis of the disease, it was aimed to develop deep convolutional neural networks (CNN) for automated PD identification based on biomarkers-derived voice signals. The developed CNN methods consisted of two main stages, including data pre-processing and fine-tunning-based transfer learning steps. To train and evaluate the performance of the developed model, datasets were collected from the mPower Voice database. SqueezeNet1_1, ResNet101, and DenseNet161 architectures were retrained and evaluated to determine which architecture can classify frequency-time information most accurately. The performance results revealed that the proposed model could successfully identify the PD with an accuracy of 89.75%, sensitivity of 91.50%, and precision of 88.40% for DenseNet-161 architecture identified as the most suitable fine-tuning architecture. The results revealed that the proposed model based on transfer learning with a fine-tuning approach provides an acceptable detection of PD with an accuracy of 89.75%. The outcomes of the study confirmed that by integrating the developed model into smart electronic devices, it will be able to develop alternative pre-diagnosis methods and will assist the physicians for PD detection during the in-clinic assessment. The success of the proposed model would imply an enhancement in the life quality of patients and a cost reduction for the national health system.

The fluorescence lifetime of different molecular species is calculated from the measured fluorescence intensity decrease following short pulsed laser excitation, by a multi-channel fitting procedure. In a FRET (Forster Resonant Energy Transfer) experiment the time dependent behaviour of the donor profile is assumed in a first view mono-exponential and the acceptor decay profile is solved analytically. A global minimization fitting algorithm has increased information content than a single channel fitting routine. In a normal FRET-FLIM experiment, the efficiency of FRET is calculated only by considering the kinetics of the donor. However, as will be shown, a considerable improvement could be achieved when time-resolved and spectral-resolved techniques are simultaneously incorporated.

The fluorescence decay of a fluorophore in many cases does not show a simple monoexponential profile. A very complex situation arises, when more than one compound must be analyzed. A considerable improvement of the measurement could be achieved when time-resolved and spectral-resolved techniques are simultaneously incorporated. SLIM (spectral fluorescence lifetime imaging) is a new technique, which combines both. Time-correlated single photon counting (TCSPC) enables high counting efficiency for biomedical applications. For spectral resolved detection a polychromator in the detection path together with a 16-channel multianode photomultiplier tube and appropriate TCSPC routing electronics are used as a highly sophisticated system. The various possibilities which SLIM offers to improve molecular imaging in living cells will be discussed as well as successfully realized applications. These include FRET (resonant energy transfer) measurements for protein interactions, related to Alzheimer's disease. Special attention will be focused on molecules involved in the processing and trafficking of the amyloid precursor protein (APP), as trafficking proteins of the GGA family and β-secretase (BACE). Taking into account also the lifetime of the acceptor could enhance reliability of the FRET result.

Abeta generation is caused by sequential cleavage of the amyloid precursor protein (APP) by two proteases, beta-site of APP cleaving-enzyme (BACE) and Gamma-secretase. Complex transport including internalization and return to the surface is necessary for this process. GGA1, a transport adaptor of the Golgi-localized gamma-ear-containing ARF-binding (GGA) family, has been shown to interact with BACE via its VHS-domain. We analyzed the differential roles of GGA1, GGA2 and GGA3 upon co-localization and interaction with BACE and effects upon APP-processing. In addition we tested the hypothesis that serine-phosphorylation of GGA1 and GGA3 affects BACE-interaction. We applied confocal imaging and fluorescence lifetime imaging microscopy (FLIM) for colocalization and interaction studies, an electrochemiluminescence-based assay to measure the influence of all three GGA upon APP processing, westernblotting to measure intracellular levels of APP cleavage fragments and insitu-hybridization to visualize correlated expression of GGAs and BACE in rat brain. All three GGAs colocalize with BACE1 at perinuclear compartments. FLIM revealed a donor-lifetime decrease indicating interaction between all three GGAs and BACE. Control experiments with GGA and BACE mutants show that the VHS domain of the GGA proteins and the DXXLL-motive in the BACE protein are necessary for this interaction. Mutants of GGA1 and GGA3 which represent non-phosphorylated forms of these proteins showed decreased lifetime whereas autoinhibited pseudo-phosphorylated mutants reversed this. Elisa and Westernblotting revealed an increase of intracellular sAPP upon overexpression of any GGA. However a decrease in sAPP secretion was observed. This effect was neither reversible with Δ-VHS mutants nor with GGA1/3 phosphorylation-mutants.Insitu-hybridization revealed spatial and time correlated expression of GGAs and BACE in postnatal and adult rats. These results indicate that all GGAs have related functions on BACE interaction, controlled by concentration, location and phosphorylation. Beside the interaction with BACE, we suggest additional BACE-independent influence of GGAs on APP transport and processing, and therefore an essential role in APP cleavage and subsequent Abeta generation.

Presbyopia is a wide spread phenomenon in elder people and is caused by the hardening of the lens in human eyes. Research is performed to make such lenses again more flexible by application of geometrically optimised cuts through the lens with a femtosecond-laser. Different protein agglomerations are responsible for the flexibility reduction of the lens. Two-photon excited fluorescence of the lens can be used as a diagnostic tool to localise such protein accumulations. In in-vitro experiments with human cataract lenses and also lenses of the Philly-mouse it could be demonstrated that with age the fluorescence increases as presbyopia proceeds. The distribution of the fluorescing compounds are not homogeneous but rather cloudy. Discrimination of the compounds by fluorescence lifetime measurements in relation of the depth in the lens is possible.

Spectral fluorescence lifetime imaging (SLIM) is an advanced imaging technique, which combines spectral with time resolved detection. Real spectral information is achieved by using a grating in front of a PML-array, which allows time-correlated single photon counting (TCSPC). Whereas spectrally resolved fluorescence imaging alone has a reasonable sensitivity, the specificity of fluorescence detection can be improved by considering the fluorescence lifetime. The various possibilities which SLIM offers to improve FRET (resonant energy transfer) will be discussed as well as successfully realized applications. These include FRET measurements for protein interactions, related to Alzheimer's disease. Special attention will be focused on molecules involved in the processing and trafficking of the amyloid precursor protein (APP), as trafficking proteins of the GGA family and β-secretase BACE). Taking into account also the lifetime of the acceptor could enhance reliability of the FRET result.

Abeta generation is caused by sequential cleavage of the amyloid precursor protein (APP) by two proteases, first beta-site of APP-cleaving enzyme (BACE) followed by Gamma-secretase. Abeta accumulates in senile plaques in Alzheimer's disease (AD). APP and BACE traffic together. GGA1, a member of the Golgi-localized gamma-ear-containing ARF binding (GGA) protein family interacts with BACE phosphorylation dependent via its VHS domain in the Golgi and influences its subcellular distribution.Therefore we were interested in the question whether GGA2 or GGA3 or both also colocalize and interact with BACE and alter its subcellular occurrence and distribution. We applied confocal imaging and a novel technique to show close protein-protein vicinity in living cells with fluorescence protein tags: spectral fluorescence lifetime imaging microscopy (FLIM). Spectral FLIM is a novel technique, which combines spectral resolved and time resolved detection. Additionally an electrochemoluminescence-based assay was established to measure the influence of GGA1, 2 and 3 upon APP processing. We found colocalization of GGA1, GGA2 and GGA3 with BACE1 at perinuclear compartments. The colocalization of GGA2 and BACE1 was enhanced in endosomal as well as Golgi structures whereas the colocalizations of GGA3 and BACE1 were smaller and could only be shown at the ER or Golgi. Regarding the GGAs and BACE FLIM we found a clear decrease in donor lifetime indicating interaction between all three GGAs and BACE. By performing control experiments with deletion mutants of GGAs and BACE we were able to show that the VHS domain of the GGA proteins and the DXXLL-motive in the BACE protein are responsible for the interaction. We observed reduced sAPP secretion upon GGA overexpression. This effect seems not to be reversible with VHS-deletion mutants of the GGAs. Therefore we suggest a BACE-independent influence of GGA1, 2 and 3 upon the APP transport and processing. These results indicate that all GGAs may have differential impact upon the transport of BACE1 and APP and may therefore play an essential role in APP cleavage and the subsequent Abeta generation.

Abeta generation is caused by sequential cleavage of the amyloid precursor protein (APP). Beta-site of APP-cleaving enzyme (BACE) is known to cleave APP followed by Gamma-secretase. The resulting Abeta accumulates in extracellular senile plaques and is associated with further neuron degeneration. APP and BACE traffic together. GGA1, a member of the Golgi-localized gamma-ear-containing ARF binding (GGA) protein family interacts with BACE phosphorylation dependent via its VHS domain in the Golgi and influences its subcellular distribution.

SLIM (spectral fluorescence lifetime imaging) is a highly sophisticated new technique, which combines spectral resolved and time resolved detection. Real spectral information is achieved by using a grating in front of a PML-array, which allows time-correlated single photon counting (TCSPC). Whereas spectrally resolved fluorescence imaging alone has a reasonable sensitivity, the specificity of fluorescence detection can be improved by considering the fluorescence lifetime.SLIM was realized on the basis of a laser scanning microscope. The fluorescence light from the second descanned detection channel was coupled into a 600 mu m multimode fibre. The end of the fibre was put into the input focal plane of an MS125 spectrograph (grating of 600 lines/mm). A PML-16 multichannel PMT module, containing a 16 channel multi-anode PMT and the TCSPC routing electronics was attached to the output of the spectrograph. The grating yields a 200 run spectral range spread over the 16 channels of the detector. The spectral bandwith of the PMT channels was about 12 nm. For fluorescence excitation, a Ti:Sa laser or alternatively a ps diode laser was used.The various possibilities which SLIM offers to improve cell diagnosis will be discussed as well as successfully realized applications. These include cancer diagnosis with endogenous and exogenous fluorophores, and FRET measurements for multiple protein interactions.

Although during the last years, significant progress was made in cancer diagnosis, using either intrinsic or specially designed fluorophores, still problems exist, due to difficulties in spectral separation of highly overlapping probes or in lack of specificity. Many of the problems could be circumvented by focusing on time-resolved methods. In combination with spectral resolved detection (spectral fluorescence lifetime imaging, SLIM) highly sophisticated fluorescence lifetime imaging can be performed which might improve specificity of cell diagnosis. To record lifetime images (τ-mapping) with spectral resolution a setup was realized consisting of a laser scanning microscope equipped with a 16 channel array for time-correlated single photon counting (TCSPC) and a spectrograph in front of the array. A Ti:Saphir laser can be used for excitation or alternatively ps diode lasers. With this system the time- and spectral-resolved fluorescence characteristics of different fluorophores were investigated in solution and in cell culture. As an example, not only the mitochondria staining dye rhodamine 123 could be easily distinguished from DAPI, which intercalates into nucleic acids, but also different binding sites of DAPI. This was proved by the appearance of different lifetime components within different spectral channels. Another example is Photofrin, a photosensitizer which is approved for bladder cancer and for palliative lung and esophageal cancer in 20 countries, including the United States, Canada and many European countries. Photofrin is a complex mixture of different monomeric and aggregated porphyrins. The phototoxic efficiency during photodynamic therapy (PDT) seems to be correlated with the relative amounts of monomers and aggregates. With SLIM different lifetimes could be attributed to various, spectrally highly overlapping compounds. In addition, a detailed analysis of the autofluorescence by SLIM could explain changes of mitochondrial metabolism during Photofrin-PDT.

Although during the last years, significant progress was made in cancer diagnosis, using either intrinsic or specially designed fluorophores, still problems exist, due to difficulties in spectral separation of highly overlapping probes or in lack of specificity. Many of the problems could be circumvented by focusing on time-resolved methods. In combination with spectral resolved detection (spectral fluorescence lifetime imaging, SLIM) highly sophisticated fluorescence lifetime imaging can be performed which might improve specificity of cell diagnosis. To record lifetime images (τ-mapping) with spectral resolution a setup was realized consisting on a laser scanning microscope equipped with a 16 channel array for time- correlated single photon counting (TCSPC) and a spectrograph in front of the array (1). A Ti:Saphir laser can be used for excitation or alternatively ps diode lasers. With this system the time- and spectral-resolved fluorescence characteristics of different fluorophores were investigated in solution and in cell culture. As an example, not only the mitochondria staining dye rhodamine 123 could be easily distinguished from DAPI, which intercalates into nucleic acids, but also different binding sites of DAPI. This was proved by the appearance of different lifetime components within different spectral channels. Another example is Photofrin, a photosensitizer which is approved for bladder cancer and for palliative lung and esophageal cancer in 20 countries, including the United States, Canada and many European countries. Photofrin is a complex mixture of different monomeric and aggregated porphyrins. The phototoxic efficiency during photodynamic therapy (PDT) seems to be correlated with the relative amounts of monomers and aggregates. With SLIM different lifetimes could be attributed to various, spectrally highly overlapping compounds. In addition, a detailed analysis of the autofluorescence by SLIM could explain changes of mitochondrial metabolism during Photofrin-PDT.

Various problems arising during molecular imaging of different fluoroprobes and metabolites used in photodynamic therapy (PDT) could be circumvented by focusing on time-resolved detection. For this, an interesting new method seems to be time-correlated single photon counting, where a time-to-amplitude converter determines the temporal position and a scanning interface connected to the scanning unit of a laser microscope determines the spatial location of a signal. In combination with spectral resolved detection (spectral lifetime imaging) the set-up achieves the features of highly sophisticated lifetime imaging systems.

Various problems arising during molecular imaging of different fluoroprobes and metabolites used in photodynamic therapy could be circumvented by focusing on time-resolved detection. For this, an interesting new method seems to be time-correlated single photon counting, where a time-to-amplitude converter determines the temporal position and a scanning interface connected to the scanning unit of a laser microscope determines the spatial location of a signal. In combination with spectral resolved detection (spectral lifetime imaging) the set-up achieves the features of highly sophisticated lifetime imaging systems.The photoactive substance on which 5-ALA PDT is based, is protoporphyrine IX which is synthesized in mitochondria. Alternatively, other metabolites from 5-ALA could be involved. Subcellular differentiation of those metabolites without extensive extraction procedures is not trivial, because of highly overlapping spectral properties. Measuring the fluorescence lifetime on a subcellular level could be a successful alternative.To record lifetime images (tau-mapping) a setup consisting on a laser scanning microscope equipped with detection units for time-correlated single photon counting and ps diode lasers for short-pulsed excitation was implemented. The time-resolved fluorescence characteristics of 5-ALA metabolites were investigated in solution and in cell culture. The lifetimes were best fitted by a biexponential fitting routine. Different lifetimes could be found in different cell compartments. During illumination, the lifetimes decreased significantly. Different metabolites of 5-ALA could be correlated with different fluorescence lifetimes. In addition cells were coincubated with the nuclear staining dye DAPI, in order to investigate the cell cycle. Using appropriate filtering or alternatively spectral lifetime imaging the time-resolved fluorescence of DAPI could be very well distinguished from 5-ALA-metabolites. In contrast to ALA, the lifetime of DAPI, which was best fitted monoexponentially did not change during photobleaching, making this dye a perfect internal standard.

A time-correlated single photon counting (TCSPC) module (SPC-730, Becker & Hickl, Germany) was connected to a laser scanning microscope (Zeiss, Germany) equipped with an ultrafast photomultiplier. Short pulse excitation was achieved with two laser diodes emitting at 398nm and 434nm with a pulse duration of 70ps and 60 ps (PicoQuant, Germany) to allow intracellular fluorescence lifetime imaging (FLIM). With this setup, fluorescence lifetime of the mitochondrial marker Rhodamine 123 could be studied in solution under the same instrumental conditions as used for fluorescence lifetime imaging of cell monolayers. With the same set of parameters, fluorescence lifetime of Rhodamine 123 was calculated with good reproducibility in mitochondria of living cells. We present here a comparison of different fitting routines, including a multiexponential fitting based on the method of Laplace transformation. Fluorescence lifetimes calculated with the multiexponential fitting routine proved to be particularly useful to study the distribution of 5-ALA metabolites in cell monolayers.

A setup consisting on a laser scanning microscope equipped with appropriate detection units was developed for time-resolved intracellular fluorescence spectroscopy and fluorescence lifetime imaging (FLIM) for on-line detection of structural changes of various biomolecules. Short-pulsed excitation was performed with a diode laser which emits pulses at 398 nm with 70 ps duration. The laser was coupled to the laser scanning microscope. For time resolved spectroscopy a setup consisting on a Czerny Turner spectrometer and a MCP-gated and -intensified CCD camera was used. Time-gated spectra within the cells were acquired by placing the laser beam in "spot scan" mode. In addition, a time-correlated single photon counting module was used to determine the fluorescence lifetime from single spots and to record lifetime images (tau-mapping). The time-resolved fluorescence characteristics of 5-ALA (5-aminolevulinic-acid), as well as 5-ALAhe (5-aminolevulinic-acid-hexylester)- induced protoporphyrine IX (PPIX) were investigated before and during PDT with subcellular resolution. For cells which were incubated with 5-ALA, a component with a fluorescence lifetime of about 7 ns was correlated with a structured fluorescence, which probably coincides with mitochondria, whereas a shorter lifetime was found in the cytoplasm. In the case of 5-ALAhe the lifetime of PPIX was longer, which could be due to different localization. During PDT the component with the longer lifetime completely vanished, whereas the shorter lifetime was retained. It seems that FLIM is a valuable method to selectively identify and localize the photodynamically active photosensitizer.

A setup consisting on a laser scanning microscope equipped with appropriate detection units was developed for time-resolved intracellular fluorescence spectroscopy and fluorescence lifetime imaging (FLIM) for online detection of structural changes of various biomolecules. Short-pulsed excitation was performed with a diode laser which emits pulses at 398 nm with 70 ps duration. The laser was coupled to the laser scanning microscope. For time resolved spectroscopy a setup consisting on a Czerny Turner spectrometer and a MCP-gated and -intensified CCD camera was used. Time-gated spectra within the cells were acquired by placing the laser beam in "spot scan" mode. In addition, a time-correlated single photon counting module (TCSPC) was used to determine the fluorescence lifetime from single spots and to record lifetime images (τ-mapping). To prove and calibrate the system, the time-resolved fluorescence characteristics of the mitochondrial marker Rhodamine 123 and 5-ALA (5-aminolevulinic-acid), as well as 5-ALAhe (5-aminolevulinic-acidhexylester)- induced protoporphyrine IX (PPIX) were investigated in solution and in cell culture. Different lifetimes could be found in different cell compartiments. During illumination, the lifetimes decreased significantly. From photobleaching experiments different metabolites of 5-ALA could be correlated with different fluorescence lifetimes. In conclusion FLIM, using ps diode lasers and TCSPC techniques is a valuable method to selectively identify and localize various metabolites of fluorescent probes during laser scanning microscopy.

A setup consisting on a laser scanning microscope (LSM 410, Zeiss, Germany) equipped with appropriate detection units was developed for time-resolved intracellular fluorescence spectroscopy and fluorescence lifetime imaging (FLIM) for on-line detection of structural changes of various biomolecules. Short-pulsed excitation was performed with a diode laser which emits pulses at 398 nm with 70 ps duration (LDH-C 400, PicoQuant, Germany). The laser was coupled to the laser scanning microscope. For time resolved spectroscopy a setup consisting on a Czerny Turner spectrometer and a MCP-gated and -intensified CCD camera was used. Time-gated spectra within the cells were acquired by placing the laser beam in “spot scan” mode. In addition, a time-correlated single photon counting module (SPC-730, Becker & Hickl, Germany) was used to determine the fluorescence lifetime of different photosensitizers from single spots and to record lifetime images (τ-mapping) [1].