Konfokales Zwei-Photonen Fluoreszenz Lifetime Laserscanning-Mikroskop
Final Report Abstract
Time-Resolved Autofluorescence Imaging of Human Donor Retina: Time and spectrally resolved measurements of autofluorescence have the potential to monitor metabolism at the cellular level. Fluorophores that emit with the same fluorescence intensity can be discriminated from each other by decay time of fluorescence intensity after pulsed excitation. We performed time-resolved autofluorescence measurements on fundus samples from a donor with significant extramacular drusen. Tissue sections from two human donors were prepared and imaged with a laser scanning microscope. The sample was excited with a titanium-sapphire laser, which was tuned to 860 nm, and frequency doubled by a BBO crystal to 430 nm. The timeresolved autofluorescence was recorded simultaneously in 16 spectral channels (445-605 nm) and bi-exponentially fitted. RPE can be discriminated clearly from Bruch's membrane, drusen, and choroidal connective tissue by fluorescence lifetime. In RPE, bright fluorescence of lipofuscin could be detected with a maximum at 510 nm and extending beyond 600 nm. The lifetime was 385 ps. Different types of drusen were found. Most of them did not contain lipofuscin and exhibited a weak fluorescence, with a maximum at 470 nm. The lifetime was 1785 picoseconds (ps). Also, brightly emitting lesions, presumably representing basal laminar deposits, with fluorescence lifetimes longer than those recorded in RPE could be detected. The demonstrated differentiation of fluorescent structures by their fluorescence decay time provides an important reference for interpretation of in vivo measurements by the new fluorescence lifetime imaging ophthalmoscopy (FLIO) on healthy subjects as well as on patients. Depth-resolved ocular fundus autofluorescence spectra and lifetimes: Two-photon excited fluorescence (TPEF) imaging of ocular tissue has recently become a promising tool in ophthalmology for diagnostic and research purposes. The feasibility and the advantages of TPEF imaging, namely deeper tissue penetration and improved high-resolution imaging of microstructures, have been demonstrated lately using human ocular samples. The autofluorescence properties of endogenous fluorophores in ocular fundus tissue are well known from spectrophotometric analysis. But fluorophores, especially when it comes to fluorescence lifetime, typically display a dependence of their fluorescence properties on local environmental parameters. Hence, a more detailed investigation of ocular fundus autofluorescence ideally in vivo is of utmost interest. The aim of this study is to determine depth-resolved the stationary and timeresolved fluorescence properties of endogenous fluorophores in ex vivo porcine ocular fundus samples by means of two-photon excited fluorescence spectrum and lifetime imaging microscopy (FSIM/FLIM). By our first results, we characterized the autofluorescence of individual anatomical structures of porcine retina samples excited at 760 nm. The fluorescence properties determined in all individual retinal layers provide a more precise characterization of the fluorescence properties of endogenous fluorophores as compared to a present in vivo approach by fluorescence lifetime imaging ophthalmoscopy (FLIO) and thereby built a foundation for an improved interpretation of pathological changes in vivo. (Diese Arbeiten wurden auf der DOG 2011 mit zwei Posterpreisen ausgezeichnet.) ABC transporter in active defense of leaf beetle larvae: Chrysomelid leaf beetles use chemical defenses to avert predatory attacks and microbial infestations. Larvae of Chrysomela populi feed on Salicaceae plants and sequester salicin. This phenolic glucoside is transported intact into the reservoirs of the larvae’s exocrine defensive glands for final transformations resulting in a deterrent chemical cocktail, released by the larvae in case of an attack by a predator. Within this sequestration process we demonstrated that an ABCC4-like transporter (CpMRP – Chrysomela populi multidrug resistance-associated protein) plays a crucial role. To probe sub-cellular localization, we carried immunohistochemical studies via two-photon excited fluorescence microscopy detection. CpMRP is localized exclusively in secretory cells with a distinct reticular pattern based on intracellular storage vesicles. Most interestingly, RNAi-silencing of CpMRP in vivo generates a defenseless phenotype incapable of secreting the defensive compounds which strongly indicates its key role in the secretion process. CpMRP seem to act as a gatekeeper while combining sequestration, secretion and therefore active defense processes. The successful, complementary combination of CpMRP, an active ABC transporter with a broad substrate spectrum, and the filter function of a still unknown selective transport process in the outer membrane of secretory cells have probably allowed many leaf beetle species to effectively adapt and coevolve with plants that offer appropriate glucoside precursors.
Publications
- A novel non-contact retina camera for the rat and its application to dynamic retinal vessel analysis. Biomedical Optics Express, 2(11):3094-3108, 2011
Link,D., Strohmaier,C., Seifert,B.U., Riemer,T., Reitsamer,H.A., Haueisen,J., Vilser, W.
- Two-photon excited fluorescence microscopy application for ex vivo investigation of ocular fundus samples. SPIE Proceeding 8086 (2011) 808605-10
Sven Peters, Martin Hammer, and Dietrich Schweitzer
- Time-Resolved Autofluorescence Imaging of Human Donor Retina Tissue from Donors with Significant Extramacular Drusen. Invest. Ophthalmol. Vis. Sci 53 (2012), 3376-86
Dietrich Schweitzer, Elizabeth R. Gaillard, James Dillon, Robert F. Mullins, Stephen Russell, Birgit Hoffmann, Sven Peters, Martin Hammer, and Christoph Biskup
