Frank Dolp

Spectrally resolved fluorescence lifetime imaging microscopy: Förster resonant energy transfer global analysis with a one- and two-exponential donor model

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 Alzheimers 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.

FLIM and SLIM for molecular imaging in PDT

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 (τ-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.

Multiwavelength FLIM: new applications and algorithms

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.

SLIM: multispectral FLIM with wide applications in cell biology

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 Alzheimers 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.

Investigation of alternative binding events between the Golgi-localized, gamma ear-containing, ARF-binding (GGA) protein family and BACE1 and its influence on the processing of amyloid precursor protein (APP)

the microarray analysis, we surveyed the binding sites of transcription factors, using the database produced by UCSC Genome Bioinformatics. Results: The increase in the DNA-binding activity of NF-kappa B and AP-1 after exposure to Abeta fibrils was suppressed by PARP-1 inhibitor, 1,5-dihydroxyisoquinoline (DHIQ), and also PARP-1 siRNA. The microarray analysis demonstrated that among 31,042 probes used, 345 and 224 probe sets showed up-regulation and down-regulation, respectively, in the astrocytes after exposure to Abeta fibrils. Furthermore, 87 probe sets showed down-regulation, while only three probe sets showed up-regulation, after addition of DHIQ. Upstream and downstream of the genes detected by these probe sets, the DNA-binding sites of other transcription factors than NF-kappa B and AP-1 were identified. Conclusions: PARP-1 plays an important role in the change of gene expression profile of astrocytes after exposure to A beta fibrils through the activation of a variety of transcription factors. By regulation of these factors, PARP-1 inhibitors could be new therapeutic and/or neuroprotective agents for Alzheimer’s disease.

Improving fluorescence diagnosis of cancer by SLIM

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.

Imaging time-resolved fluorescence characteristics of organelle specific fluorophores and photosensitizers using ps pulsed diode lasers and TCSPC techniques

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.