Gregor Liebsch

Luminescence Lifetime Imaging of Oxygen, pH, and Carbon Dioxide Distribution Using Optical Sensors

We present a modular system for time-resolved two-dimensional luminescence lifetime imaging of planar optical chemical sensors. It is based on a fast, gateable charge-coupled device (CCD) camera without image intensifier and a pulsable light-emitting diode (LED) array as a light source. Software was developed for data acquisition with a maximum of parameter variability and for background suppression. This approach allows the operation of the system even under daylight. Optical sensors showing analyte-specific changes of their luminescence decay time were tested and used for sensing pO2, pCO2, pH, and temperature. The luminophores employed are either platinum(II)-porphyrins or ruthenium(II)-polypyridyl complexes, contained in polymer films, and can be efficiently excited by blue LEDs. The decay times of the sensor films vary from 70 μs for the Pt(II)-porphyrins to several 100 ns for the Ru(II) complexes. In a typical application, 7 mm-diameter spots of the respective optical sensor films were placed at the bottom of the wells of microtiterplates. Thus, every well represents a separate calibration chamber with an integrated sensor element. Both luminescence intensity-based and time-resolved images of the sensor spots were evaluated and compared. The combination of optical sensor technology with time-resolved imaging allows a determination of the distribution of chemical or physical parameters in heterogeneous systems and is therefore a powerful tool for screening and mapping applications.

Luminescence Lifetime Temperature Sensing Based on Sol‐Gels and Poly(acrylonitrile)s Dyed with Ruthenium Metal–Ligand Complexes

Temperature-sensitive materials based on the temperature probe ruthenium-tris-1,10-phenathroline (Ru(phen)) are presented. As its luminescence, the intensity and decay time of which are both temperature sensitive, is quenched by oxygen, Ru(phen) needs to be encapsulated in materials with very low permeability to molecular oxygen. Its incorporation into sol-gels and poly(acrylonitrile)s (PANs) is investigated here and it is shown that the resulting materials can be deposited onto optical fibers for use as temperature optodes.

In Vivo Phosphorescence Imaging of pO2 Using Planar Oxygen Sensors

Objective: Oxygen‐dependent quenching of luminescence of metal porphyrin complexes has been used to image the pO2 distribution over tumor and normal tissue.

Effects of light fractionation and different fluence rates on photodynamic therapy with 5-aminolaevulinic acid in vivo.

To improve efficacy of photodynamic therapy (PDT) with intravenously administered 5-aminolaevulinic acid (ALA) fractionating the light dose or reducing the light intensity may be a possibility. Therefore, Syrian Golden hamsters were fitted with dorsal skinfold chambers containing an amelanotic melanoma (n=26). PDT was performed (100 mW cm−2, 100 J cm−2, continuously or fractionated, and 25 mW cm−2, 100 J cm−2; continuously or fractionated) using an incoherent light source following i.v. application of ALA. Following fractionated irradiation, the light was paused after 20 J cm−2 for 15 min. Prior to and up to 24 h after PDT tissue, pO2 was measured using luminescence lifetime imaging. The efficacy was evaluated by measuring the tumour volume of amelanotic melanoma cells grown subcutaneously in the back of Syrian Golden hamsters (n=36). Only high-dose PDT resulted in a significant decrease of pO2. Irrespective of the mode of irradiation only high-dose PDT induced complete remission of all tumours (13 out of 13). It could be shown that low-dose PDT failed to induce a significant decrease of pO2. No significant effect of fractionated irradiation was shown regarding the therapeutic efficacy 28 days after PDT. Thus performing a fractionated PDT with ALA or reducing the light intensity seems not to be successful in clinical PDT according to the present data.

Dual Lifetime Referencing (DLR) - A New Scheme for Converting Fluorescence Intensity into a Frequency-Domain or Time-Domain Information

Fluorescence spectroscopy and NMR spectroscopy are probably the most powerful spectroscopies at present albeit with very different (and highly complementary) fields of application. Fluorometry can be based on the intrinsic fluorescence of (bio)molecules or ions, or on the use of fluorescent probes, indicators, or labels Numerous parameters can be measured which include intensity, decay time, polarization, radiative and non-radiative energy transfer, quenching efficiency, and combinations thereof Fluorescence microscopy and imaging are other widely applied techniques, and multi-dimensional and synchronous fluorescence spectroscopy have gained some interest in recent years.

An imaging method for oxygen distribution, respiration and photosynthesis at a microscopic level of resolution

Biological samples are far from homogeneous, with complex compartmentation being the norm. Major physiological processes such as respiration do not therefore occur in a uniform manner within most tissues, and it is currently not possible to image its gradients in living plant tissues. A compact fluorescence ratiometric-based device is presented here, consisting of an oxygen-sensitive foil and a USB (universal serial bus) microscope. The sensor foil is placed on the sample surface and, based on the localized change in fluorescence signal over time, information about the oxygen consumption (respiration) or evolution (photosynthesis) can be obtained. Using this imaging technique, it was possible to demonstrate the spatial pattern of oxygen production and consumption at a c. 20-μm level of resolution, and their visualization in the rhizosphere, stem and leaf, and within the developing seed. The oxygen mapping highlighted the vascular tissues as the major stem sink for oxygen. In the leaf, the level of spatial resolution was sufficient to visualize the gas exchange in individual stomata. We conclude that the novel sensor set-up can visualize gradients in oxygen-consuming and producing processes, thereby facilitating the study of the spatial dynamics of respiration and photosynthesis in heterogeneous plant tissues.

Transcutaneous pO2 imaging during tourniquet-induced forearm ischemia using planar optical oxygen sensors.

Background: Oxygen‐dependent quenching of luminescence using transparent planar sensor foils was shown to overcome the limitations of the polarographic electrode technique in an animal model. This method was then transferred to a clinical setting to measure the transcutaneous pO2 (ptcO2).

Multi-ion imaging using fluorescent sensors in a microtiterplate array format

A novel type of sensor array destined for water analyses is described. The sensor delivers simple on/off patterns of complex ion mixtures. Fluorescent indicators for Ca2+, Na+, Mg2+, SO42-, Cl− and Hg2+ were arranged in microtiterplates and the analytical information was imaged with a CCD camera within microseconds using an intrinsically referencing method.

Ratiometric luminescence 2D in vivo imaging and monitoring of mouse skin oxygenation

Tissue oxygenation plays a critical role in the pathogenesis of various diseases, but non-invasive, robust and user-friendly methods for its measurement in vivo still need to be established. Here, we are presenting an in vivo oxygen-detection system that uses ratiometric luminescence imaging (RLI) as a readout scheme to determine the skin oxygen tension of mouse hind footpads via side-by-side comparison with more established techniques including luminescence-lifetime imaging using planar sensor films and the polarographic electrode as the gold standard. We also demonstrate that this technology allows the detection of changes in mouse skin tissue oxygenation induced by subjecting mice to systemic hypoxia. The data demonstrate oxygen imaging based on RLI to be a most useful tool for reliably and easily analyzing and monitoring skin tissue oxygenation in vivo. This technology will advance our understanding of local regulation of skin tissue oxygenation in various disease conditions.

Low-oxygen tensions found in Salmonella-infected gut tissue boost Salmonella replication in macrophages by impairing antimicrobial activity and augmenting Salmonella virulence

In Salmonella infection, the Salmonella pathogenicity island‐2 (SPI‐2)‐encoded type three secretion system (T3SS2) is of key importance for systemic disease and survival in host cells. For instance, in the streptomycin‐pretreated mouse model SPI‐2‐dependent Salmonella replication in lamina propria CD11c−CXCR1− monocytic phagocytes/macrophages (MΦ) is required for the development of colitis. In addition, containment of intracellular Salmonella in the gut critically depends on the antimicrobial effects of the phagocyte NADPH oxidase (PHOX), and possibly type 2 nitric oxide synthase (NOS2). For both antimicrobial enzyme complexes, oxygen is an essential substrate. However, the amount of available oxygen upon enteroinvasive Salmonella infection in the gut tissue and its impact on Salmonella–MΦ interactions was unknown. Therefore, we measured the gut tissue oxygen levels in a model of Salmonella enterocolitis using luminescence two‐dimensional in vivo oxygen imaging. We found that gut tissue oxygen levels dropped from ∼78 Torr (∼11% O2) to values of ∼16 Torr (∼2% O2) during infection. Because in vivo virulence of Salmonella depends on the Salmonella survival in MΦ, Salmonella–MΦ interaction was analysed under such low oxygen values. These experiments revealed an increased intracellular replication and survival of wild‐type and t3ss2 non‐expressing Salmonella. These findings were paralleled by blunted nitric oxide and reactive oxygen species (ROS) production and reduced Salmonella ROS perception. In addition, hypoxia enhanced SPI‐2 transcription and translocation of SPI‐2‐encoded virulence protein. Neither pharmacological blockade of PHOX and NOS2 nor impairment of T3SS2 virulence function alone mimicked the effect of hypoxia on Salmonella replication under normoxic conditions. However, if t3ss2 non‐expressing Salmonella were used, hypoxia did not further enhance Salmonella recovery in a PHOX and NOS2‐deficient situation. Hence, these data suggest that hypoxia‐induced impairment of antimicrobial activity and Salmonella virulence cooperate to allow for enhanced Salmonella replication in MΦ.