Rudolf W. Steiner

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.

Photodynamic tumor therapy and on-line fluorescence spectroscopy after ALA administration using 633-nm light as therapeutic and fluorescence excitation radiation

Photodynamic therapy (PDT) and on-line fluorescence spectroscopy were carried out on human tumors after 5-aminolevulinic acid (ALA) administration using 633-nm light of a dye laser as therapeutic radiation and as fluorescence excitation radiation. This has the advantages of (1) enabling use of one laser for PDT and fluorescence diagnosis only, (2) enabling the possibility of on-line fluorescence measurements, and (3) exciting protoporphyrin molecules in deep tissue layers. Monte Carlo calculations were carried out to determine excitation and fluorescence photon distribution in case of red and violet excitation radiation. The results show the possibility of depth-resolved measurements on the fluorophore distribution by variation of excitation wavelength. The high penetration depth of 633-nm radiation results in a higher ratio of the 700-nm protoporphyrin fluorescence of the xenotransplanted tumor It to the skin Is compared with 407-nm excitation. No values greater than 1 for the ratio It/Is were found in the case of intravenous ALA injection even for red excitation. Therefore, a large amount of ALA will be metabolized in the skin and can cause photosensitivity of the patient when applied systematically. In contrast, protoporphyrin fluorescence limited to the pretreated skin area was detected in case of topically applied ALA to patients with mycosis funcoides and erythroplasy of Queyrat. The influence of remitted excitation light and of the spontaneous radiation from the laser as well as the possible excitation of food-based degradation products of chlorophyll has to be considered in high-sensitivity fluorescence measurements.

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.

Short-pulsed diode lasers as an excitation source for time-resolved fluorescence applications and confocal laser scanning microscopy in PDT

This article describes a setup for subcellular time-resolved fluorescence spectroscopy and fluorescence lifetime measurements using a confocal laser scanning microscope in combination with a short pulsed diode laser for fluorescence excitation and specimen illumination. The diode laser emits pulses at 398 nm wavelength with 70 ps full width at half maximum (FWHM) duration. The diode laser can be run at a pulse repetition rate of 40 MHz down to single shot mode. For time resolved spectroscopy a spectrometer setup consisting of an Czerny Turner spectrometer and a MCP-gated and -intensified CCD camera was used. Subcellular fluorescence lifetime measurements were achieved using a time-correlated single photon counting (TCSPC) module instead of the spectrometer setup. The capability of the short pulsed diode laser for fluorescence imaging, fluorescence lifetime measurements and time-resolved spectroscopy in combination with laser scanning microscopy is demonstrated by fluorescence analysis of several photosensitizers on a single cell level.

Investigation of multilayered tissue with in vivo reflectance measurements

Light propagation in multi-layered tissues was studied theoretically and experimentally on a rabbit thigh and on human forearms. Theoretical investigations were based on a special solution of the diffusion equation considering layered materials or on Monte Carlo simulations. The spatially resolved reflectance of a small diameter laser beam (633 or 751 nm) incident on the tissue was measured in vivo with a CCD-camera based system previously tested with homogeneous tissue simulating phantoms. The optical coefficients were determined either by non-linear regressions to a solution of the diffusion equation or to Monte Carlo simulations. We fitted up to four independent parameters to describe the spatially resolved reflectance from two- and three-layered tissues.

Light penetration in human skin: in-vivo measurements using isotropic detector

An isotropic probe for direct measurement of fluence rate in tissue was made by melting 110 micrometers -core fiber to less than 0.4 mm sphere and painting by scattering material. Anisotropy response for the best probe of 0.35 mm diameter was +/- 10% in angular range to +/- 150 degree(s). The general position error of the probe in the tissue provided by experimental setup was less than 0.1 mm. The fluence rate was measured in the human leg in vivo at a depth of up to 5 mm for incident irradiance of 100 mW/cm2 and wavelength 625 nm (dye laser) for beam diameters of 1 mm and 5 mm. Effective attenuation coefficients of about 0.6 mm-1 (for depth of up to 1 mm) and 0.7 mm-1 (for muscle layers) were obtained. Differences in light distribution in the skin comparative to the deeper layers were not found.

Laser-induced autofluorescence of cells and tissue

The laser induced in-vivo autofluorescence in the visible spectral region of cells and tissues was studied using stationary and time-resolved fluorescence measurements as well as video- intensified- and confocal laser-scanning microscopy. Due to their excitation and emission spectra and their decay kinetics (ps- and ns-region) at least three intrinsic fluorophores were so far differentiated and attributed to the reduced pyridine coenzymes NADH/NADPH, free and bound flavin molecules and endogenous porphyrins. It was found, that defects in the mitochondrial respiratory chain as well as photodynamically or chemically induced cell destruction results in an increase of the fluorescence intensity in the blue/green spectral range.

Photodynamic tumor therapy and on-line fluorescence spectroscopy after aminolevulinic acid administration using 633-nm light as therapeutic and fluorescence excitation radiation

PDT and on-line fluorescence spectroscopy were carried out on human tumors after ALA- administration using 633 nm-light of a dye laser as therapeutic radiation and as fluorescence excitation radiation. This has the following advantages: (1) use of one laser for PDT and fluorescence diagnosis only, (2) the possibility of on-line fluorescence measurements, and (3) excitation of protoporphyrin molecules in deep tissue layers. Monte Carlo calculations were carried out to determine the excitation and fluorescence photon distribution in the case of red and violet excitation radiation. The results show the possibility of depth-resolved measurements on the fluorophore distribution by variation of the excitation wavelength. The influence of remitted excitation light and of the spontaneous radiation from the laser as well as the possible excitation of food-based degradation products of chlorophyll has to be considered in high-sensitive fluorescence measurements.

Determination of the optical properties of tissue by spatially resolved transmission measurements and Monte Carlo simulations

The spatially resolved transmission through porcine liver is measured with a CCD-Camera. A solution of the diffusion equation is derived to investigate whether the optical coefficients can be deduced from this experimental geometry. The inverse problem is solved with a nonlinear regression program using Monte Carlo simulations as solutions of the forward problem. The experiment is tested with latex-spheres, which are described by Mie-Theory.

Fast OCT assembly using wide-area detection

OCT with wide area detection has proven to be a good instrument to obtain tomographic images of epithelial structures. A depth of 2 mm was reached with a CCD-camera setup. Due to the lack of an effective image preprocessing the data acquisition time was too long to carry out measurements in vivo. A new detector head using line detector arrays was developed with the advantage of interference detection by complementary detectors and data reduction by analog computing.