Herbert Schneckenburger

In-vivo fluorescence detection and imaging of porphyrin-producing bacteria in the human skin and in the oral cavity for diagnosis of acne vulgaris, caries, and squamous cell carcinoma

Certain bacteria are able to synthesize metal-free fluorescent porphyrins and can therefore be detected by sensitive autofluorescence measurements in the red spectral region. The porphyrin-producing bacterium Propionibacterium acnes, which is involved in the pathogenesis of acne vulgaris, was localized in human skin. Spectrally resolved fluorescence images of bacteria distribution in the face were obtained by a slow-scan CCD camera combined with a tunable liquid crystal filter. The structured autofluorescence of dental caries and dental plaque in the red is caused by oral bacteria, like Bacteroides or Actinomyces odontolyticus. `Caries images were created by time-gated imaging in the ns-region after ultrashort laser excitation. Time-gated measurements allow the suppression of backscattered light and non-porphyrin autofluorescence. Biopsies of oral squamous cell carcinoma exhibited red autofluorescence in necrotic regions and high concentrations of the porphyrin-producing bacterium Pseudomonas aerigunosa. These studies suggest that the temporal and spectral characteristics of bacterial autofluorescence can be used in the diagnosis and treatment of a variety of diseases.

Test system for human tumor cell sensitivity to drugs on chicken chorioallantoic membranes.

Dear Editor: Cultured cells probably lose part of their natural cell functions if they are not in close contact with each other in a tissue connection. To observe their behavior it was necessary to isolate them from their natural environment and force them to survive in an artificial one. This report describes a new microscopic technique which allows simultaneous study of tumor cells and normal tissue cells growing together in the same tissue preparation. A human tumor was transplanted for the first time in 1912 on the chorioallantoic membrane (CAM) of a chick embryo (Murphy et al., J. Am. Med. Assoc. 59 :874-875; 1912). Our preparation of the eggs is a modification of a previously described technique (Auerbach et al., Devl. Biol. 41 :391-394; 1974). In fertilized eggs, the CAM gets exposed by cutting a round window of about 20 mm in diameter in the egg shell. HT 29 rectum carcinoma cells are seeded into the center of a small silicon ring (2 mm in diameter) onto the CAM. The eggs are incubated for another 3 days at 37 ° C in a humidified incubator. Then the CAM in and around the silicon ring is isolated and transferred into a close perfusion chamber (POC), operating under continuous perfusion of a 0.9% NaC1 solution. This preparation keeps the cells alive up to 4 hours and allows their observation by video enhanced microscopy. Fig. 1 shows the morphology of a through focus picture of the CAM tissue. Tumor cells grown on the normal CAM cells are shown in Fig. 2. To demonstrate the selective accumulation of a photosensitizer [meso-tetra (4N-methylpyridyl-photoporphyrin) (T4MPyP)] in the tumor cells (Fig. 3), the dye is either injected into a vein of the CAM or perfused after the CAM preparation. The technique shown here allows the observation of growing tumor cells in a natural cell formation. It enables to study changes in the tumor tissue induced by cancer therapy for several hours by means of light and fluorescence video enhanced microscopy. This technique opens a new field in the search for novel anti-cancer drugs and in chemosensitivity tests.

Fluorescence techniques in biomedical diagnostics

An overview on some advanced techniques of fluorescence spectroscopy and fluorescence microscopy is given. These techniques include time-resolved fluorescence spectroscopy, energy transfer spectroscopy (FRET), total internal reflection fluorescence microscopy (TIRFM) and fluorescence lifetime imaging (FLIM). The principle of these methods is explained, and numerous applications are described.

Time-gated spectroscopy of intrinsic fluorophores in cells and tissues

Based on novel time-resolving methods, the autofluorescence of saccharomyces, cultivated endothelial cells and biopsy specimens of human bladder was measured. The emission was found to be composed by the coenzymes NADH (free and protein-bound) and flavins with a concentration ratio of 100:1 between free NADH and flavin molecules. The fluorescence intensity of free NADH appeared to be a measure of the respiratory function. In addition, epithelial and connective tissues could be distinguished by the intensity ratio at 435 and 460 nm, which may be affected by the relation of bound and free NADH, but also by the extracellular fluorescence of elastin.

Time-resolved total internal reflection fluorescence spectroscopy: application to the membrane marker laurdan

A compact device for variable-angle total internal reflection flourescent microscopy was developed. A pulsed Nd:YAG laser operated at 355 nm was adapted using a multimode quartz fiber and collimating optics with a variable angle of incidence between 64 degrees and 72 degrees. Fluorescence spectra of BKEz-7 endothelial cells incubated with the membrane marker 6-dodecanoyl-2- dimethylamino-naphthalene were measured under TIR illumination as a function of the angle of incidence, incubation time and temperature. Emission bands around 440 nm and 490 nm were detected corresponding to laurdan locate within the gel phase or liquid crystalline phase of cellular lipids, respectively. The generalized polarization GP = (I440-I490)/(I440+I490) was used as a measure of intracellular temperature with a precision of ±1°C in the physiologically interesting range between 35°C and 38°C. Following pulsed laser excitation, the time delay between excitation and fluorescence detection was varied. A time gate at 10-15 ns after laser excitation revealed to be an optimum for spectral discrimination of the two emission bands. Fluorescence intensity IF of both bands decreased continuously when the angle of incidence Θ was increased. Between Θ = 68° and 72° the angular dependence corresponded to a fluorophore located within a thin layer (plasma membrane) at 150-200nm distance from the light reflecting surface. Between Θ=65° and 68° additional contributions from intracellular membranes were observed.

Fast photomultiplier tube gating technique for time-resolved fluorescence measurements

A nanosecond gating technique applied to conventional, off the shelf, photomultiplier tubes (PMTs) is described. This technique is most suitable for detection and analysis of fluorescence, induced by pulsed lasers. A high voltage square wave generator is applied to 3 dynodes of the PMT. The result is a fast time-gated optical detector with a high dynamic range of approximately 80 dB. The active state of the gate, i.e. the pulsewidth of the applied square wave, sets the integration time in accordance to the decay time of the fluorescence signal. It can be selected by the user from less than 2 ns to several hundred nanoseconds. The pulse generator gets its energy from a transmission line, which is charged by a DC voltage of 650 Volts. This determines the magnitude of the pulsed voltage, which is applied to the 3 PMT stages. Twice the electrical length of the transmission line gives the pulse width. The pulse is initiated by a fast electrical switch with switching times of approximately 700 ps and a low time jitter of only 200 ps related to the trigger input. A distinct separation of the fluorescence and superposing background, e.g. scattered laser light, is achieve due to fast pulse transitions. Output signals from the PMT are processed by a fast integration. This can reduce the effects of electrical cross talk of the gating pulse into the signal line to sufficiently low values.

Comparative in vitro and in vivo measurements of hydrophilic and hydrophobic porphyrins

The photosensitizers uroporphyrin III (UP III), coproporphyrin III (CP III), and protoporphyrin IX (PP IX) were examined with respect to their composition (by different molecular species), uptake and distribution in cells and tissues, intracellular pH value, cytotoxicity and formation of photoproducts. Whereas hydrophilic UP III and CP III were characterized by low cytotoxicity and preferential accumulation in lysosomes, hydrophobic PP IX was found to be rather cytotoxic and mainly localized in cellular membranes. Rapid uptake in tissues and some preferential location in tumor cells were detected for hydrophobic photosensitizers.

Studies on porphyrin photoproducts in solution, cells, and tumor tissue

Light excitation of photosensitizing porphyrins leads to cytotoxic reactions. In addition, photobleaching and photoproduct formation occur indicating photosensitizer destruction. Photoproducts from hematoporphyrin (HP) fluoresce in aqueous solution at 642 nm, whereas photoproducts from protoporphyrin (PP) in hydrophobic environment emit around 670 nm and exhibit pronounced absorption at 665 nm. Photoproduct formation depends on singlet oxygen. The photoproducts exhibit faster fluorescence decay kinetics compared with nonirradiated porphyrins, as shown by time-grated spectroscopy and fluorescence decay measurements. Photoproduct fluorescence was observed during light exposure of cells and of tumor-bearing, nude mice, following administration of Hematoporphyrin Derivative (HpD), tetramethyl-HP, and PP. Photoconversion was also detected with naturally-occurring porphyrins (PP-producing bacteria) and ALA-simulated biosynthesis of PP in tumor tissue and in skin lesions of patients (psoriasis, mycosis fungoides). The efficiency of PDT with porphyrin photoproducts was found to be low in spite of the strong electronic transitions in the red spectral region.

Total internal reflection energy transfer (TIRET) microscopy for analysis of focal adhesions in living cells

Total internal reflection fluorescence microscopy (TIRFM) is used to measure non-radiative energy transfer between membrane associated proteins in living cells. Measurements are concentrated on focal contacts and their associated proteins focal adhesion kinase (FAK) and Paxillin (Pax) which play major roles with respect to cell migration, growth, and survival. These proteins are visualized after fusion with variants of green fluorescent protein (ECFP and EYFP), and an intermolecular energy transfer ECFP -> EYFP is deduced from fluorescence spectra as well as from fluorescence decay kinetics of single cells.

In vivo uptake and biodistribution of lipophilic and hydrophilic photosensitizers

Uptake and biodistribution of photosensitizers are crucial parameters for evaluating the efficacy of photodynamic therapy (PDT). We used the tumor baring chorioallantoic membrane (CAM) as an in vivo model system to study biodistribution of hydrophilic and lipophile compounds of natural porphyrins and pthalocyanines using confocal laser scanning microscopy (LSM) and time-gated microspectrometry. Simultaneously we observed tumor vessels and tumor cells after intravascular application at different incubation times. Differences in biodistribution of hydrophilic and lipophilic sensitizers could be observed. Hydrophilic compounds were characterized by selective accumulation in tumor cells. In contrast, more lipophilic sensitizers are accumulated in endothelial cells at short incubation times as well as in tumor cells. These facts are in correlation with our studies in tumor cells and endothelial cells in vitro, where hydrophilic sensitizers are characterized by low uptake and low phototoxicity towards endothelial cells. In contrast more lipophilic sensitizers were rapidly taken up and showed high phototoxicity.