Gereon Huettmann

Model system for investigating laser-induced subcellular microeffects

Background: Laser induced protein denaturation is of fundamental interest for understanding the mechanisms of laser tissue interaction. Conjugates of nanoabsorbers coupled to proteins are presented as a model system for investigating ultrafast protein denaturation. Irradiation of the conjugates using repetitive picosecond laser pulses, which are only absorbed by the nanoabsorbers, could result in effects with a spatial confinement of less than 100 nm. Materials and Methods: Experiments were done with bovine intestinal alkaline phosphates (aP) coupled to 15 nm colloidal gold. This complex was irradiated at 527 nm wavelength and 35 ps pulse width with a varying number of pulses ranging form one up to 104. The radiant exposure per pulse was varied form 2 mJ/cm2 to 50 mJ/cm2. Denaturation was detected as a loss of protein function with the help of the fluorescence substrate 4MUP. Results and discussion: Irradiation did result in a steady decrease of the aP activity with increasing radiant exposures and increasing number of pulses. A maximal inactivation of 80% was reached with 104 pulses and 50 mJ/cm2 per pulse. The temperature in the particles and the surrounding water was calculated using Mies formulas for the absorption of the nanometer gold particles and ana analytical solution of the equations for heat diffusion. With 50 mJ/cm2, the particles are heated above the melting point of gold. Since the temperature calculations strongly depend on changes in the state of matter of the particles and water, a very sophisticated thermal model is necessary to calculate exact temperatures. It is difficult to identify one of the possible mechanisms, thermal denaturation, photochemical denaturation or formation of micro bubbles from the dependance of the inactivation on pulse energy and number of applied pulses. Therefore, experiments are needed to further elucidate the damage mechanisms. In conclusion, denaturing proteins irreversibly via nanoabsorbers using picosecond laser pulses is possible. The confinement of the heat to the nanoabsorbers when irradiating with picosecond pulses suggests that the denaturation of proteins could be possible with nanometer precision in cells with this approach. However, the mechanism of protein inactivation, which is part of present investigations, is crucial for the precision of such nanoeffects.

Slit-lamp-adapted fourier-domain OCT for anterior and posterior segments: preliminary results and comparison to time-domain OCT.

Purpose: To evaluate the diagnostic potential of a slit-lamp (SL)-adapted Fourier-domain (= spectral radar, SR) optical coherence tomography (OCT)—SL-SR-OCT—instrument as an in vivo imaging device for use in examinations of the anterior and posterior segments. Materials and Methods: In a pilot study, 88 eyes from 70 healthy volunteers and patients were examined using a prototype Fourier-domain SL-SR-OCT system. Results were compared to those from the following commercially available systems: the 1310-nm SL-OCT (Heidelberg Engineering, Heidelberg, Germany) for anterior segment and the Stratus OCT (Zeiss Meditec, Jena, Germany) for posterior segment imaging. Our SL-SR-OCT provides 1025 axial scans, 5000 Hz line-scan frequency, scan length of up to 8 mm, axial depth in air of 3.5 mm, and resolution of 9 μm. For posterior visualization, a hand-held 78-diopter ophthalmoscopic lens was used. Results: Our SL-SR-OCT system allowed simultaneous scanning with direct biomicroscopic and SL imaging of anterior and posterior segment structures. Anatomical structures and pathological changes were displayed with high resolution and excellent contrast. Measurements of corneal and retinal thickness were possible. In comparison to images obtained by the SL-OCT, our SL-SR-OCT boasted a higher resolution, thus providing more clinically relevant details of the corneal epithelium, internal structure of filtering blebs, etc. Complete imaging of the chamber angle was limited, however, due to the backscattering properties of the sclera at 830 nm. For posterior segment imaging, excellent delineation of the macula and optic nerve head details, with a distinct portrayal of macular pathology and retinal edema, was possible with SL-SR-OCT. Conclusion: SL-SR-OCT enables detailed imaging of physiological and pathological anterior and posterior segment structures. As a multi-purpose device, it offers a wide spectrum of applications, with high-quality OCT-imaging, in a comfortable setting without the need to move the patient.

Laser-generated micro- and nanoeffects: inactivation of proteins coupled to gold nanoparticles with nano- and picosecond pulses

Background: Protein denaturation in the fs-ns time regime is of fundamental interest for high precision applications in laser tissue interaction. Conjugates of colloidal gold coupled to proteins are presented as a model system for investigating ultrafast protein denaturation. It is expected that irradiation of such conjugates in tissue using pico- up to nanosecond laser pulses could result in effects with a spatial confinement in the regime of single macromolecules up to organelles. Materials and Methods: Experiments were done with bovine intestinal alkaline phosphatase (aP) coupled to 15 nm colloidal Gold. This complex was irradiated at 527 nm/ 532 nm with a variable number of pico- and nanosecond pulses. The radiant exposure per pulse was varied from 2 to 50 mJ/cm2 in the case of the picosecond pulses and 10 to 500 mJ/cm2 in the case of the nanosecond pulses. Denaturation was detected as a loss of protein function with the help of the uorescence substrate 4MUP. Results and Discussion: Irradiation did result in a steady decrease of the aP activity with increasing radiant exposures and increasing number of pulses. Inactivations up to 80% using 35 ps pulses at 527 nm with 50 mJ/cm2 and a complete inactivation induced by 16 ns pulses at 450 mJ/cm2 are discussed. The induced temperature in the particles and the surrounding water was calculated using Mies formulas for the absorption of the nanometer gold particles and an analytical solution of the equations for heat diffusion. The calculated temperatures suggest that picosecond pulses heat a molecular scaled area whereas nanosecond pulses could be used for targeting larger cellular compartiments. It is difficult to identify one of the possible damage mechanisms, i.e. thermal denaturation or formation of micro bubbles, from the dependance of the inactivation on pulse energy and number of applied pulses. Therefore experiments are needed to further elucidate the damage mechanisms. The observed inactivation dependencies on applied energy and radiant power can not be explained with one or two photon photochemistry. In conclusion, denaturing proteins irreversibly via nanoabsorbers using pico-/ nanosecond laser pulses is possible. The expected confinement of the heat to the nanoabsorbers suggests that denaturation of proteins with nanometer precision could be possible with this approach. However, the mechanism of protein inactivation, which is part of present investigations, is crucial for the precision of such nanoeffects.

Chemical instability of 5-aminolevulinic acid (ALA) in aqueous solution

Five-aminolevulinic acid (ALA) is being used to induce formation and accumulation of endogenous protoporphyrin IX both in preclinical and clinical studies on photodynamic therapy and diagnosis. At the high concentrations needed for clinical application, ALA is not stable in aqueous solutions in the neutral to basic pH range. This could be of critical importance for the clinical use of ALA. The chemical instability of ALA was studied by using UV-Vis absorption spectroscopy. Our results show that ALA undergoes a chemical reaction, which is a nonreversible condensation process yielding two different products -- a hydropyrazine and a pyrazine, the latter formed by oxidation of the hydropyrazine. Preliminary kinetic studies of this reaction showed a complex pH-, concentration and temperature-dependency of the reaction rate. On the basis of these studies we optimized conditions for the preparation of ALA solutions to be used in clinical trials, leading to the expected accumulation of protoporphyrin IX in tumor cells.

Protoporphyrin IX distribution after intra-articular and systemic application of 5-aminolevulinic acid in healthy and arthritic joints

Arthroscopic synovectomy, which is limited today to the large joints, is an important early treatment of rheumatoid arthritis (RA). Photodynamic therapy (PDT) is potentially to be a less invasive method of removing the synovial membrane. Therefore, in a rabbit model of RA, the accumulation of the photosensitizer Protoporphyrin IX (PPIX) after intra-articular and systemic application of ALA into arthritic rabbit knee joints was studied in skin, patella, synovial tissue, and meniscus by fluorescence microscopy. PPIX fluorescence was measured in biopsies taken at different times after application of neutral and acid ALA solutions. Significant PPIX fluorescence was observed in the synovial membrane and skin 2 and 4 hours after application. Using intra-articular application, ALA solutions prepared with pH 5.5 were at least as efficient as neutral solutions in sensitizing the synovial membrane. Skin also showed PPIX within 4 hours after application. After 24 hours, a marginal PPIX fluorescence was detected in these tissues. On the other hand, in cartilage and meniscus significant PPIX accumulation was still observed 24 hours after ALA injection. Systemic application of ALA also showed a good accumulation of PPIX. Further experiments are needed to show whether accumulation of the photosensitizer and tissue selectivity are sufficient for a successful treatment of rheumatoid synovitis.

Fluorescence microscopy studies on ALA-sensitized tissues

Fluorescence microscopy has the potential to study the spatial distribution of photosensitizers in tissue samples with cellular or subcellular resolution. A fluorescence microscope was developed to study the distribution of photosensitizer in tissue samples by acquiring fluorescence images in various spectral ranges and spatially resolved fluorescence spectra both from identical samples. Both methods provide complementary information, since the fluorescence images show the distribution of the sensitizers with a high spatial resolution whereas spatially resolved fluorescence spectra can identify the sensitizers and separate their fluorescence from background light emission by the spectral shape of the fluorescence. Protoporphyrin IX (PPIX) distribution induced by 5-aminolevulinic acid (ALA) was studied by fluorescence microscopy in basal cell carcinoma (BCC) and in cervical intraepithelial neoplasia (CIN). In an attempt to understand the varying success in treating BCC with topically applied ALA the PPIX distribution was studied in BCC samples of 10 patients. A strong fluorescence was observed in tumor cells as well as in epidermis, sebaceous glands, and hair follicles. The depth of PPIX sensitization of the BCCs ranged from 0.4 to 3 mm and the ratio of tumor versus epidermal fluorescence of uninvolved skin was near one. In the BCCs an uneven sensitization with a lower fluorescence in the center of the tumor was often observed. Samples of the cervical mucosa also showed PPIX fluorescence in the endothelial layer, the malignant tissues and the glands. No increased fluorescence of the dysplastic lesions compared to the epithelium was observed.

Measurement of optical-transport coefficients of Intralipid in visible and NIR range

This article presents a modified method of measuring the optical-transport-coefficients of intralipid: the diffuse reflectance of intralipid with added-ink Rdink is measured by using an integrating sphere to calculate the scattering coefficient (mu) s, the effective attenuation coefficient (mu) eff is measured by scanning the surface of pure intralipid suspension with a cut-end, high NA fiber-optic tip ((phi) 600micrometers , NA equals 0.48) in order to directly derive the absorption (mu) a of pure intralipid. In the same way, their wavelength dependencies between 0.48 - 0.85 micrometers are measured by utilizing Ar+, dye and Ti:Sapphire lasers. Experiments show that (mu) s((lambda) ) varies with (lambda) according to the previously reported Mie theory, (mu) s((lambda) ) decreases with (lambda) while Rdink((lambda) ) is nearly invariant within the wavelength range; the scattering anisotropy g((lambda) ) tends to decrease linearly with (lambda) from 0.91 to 0.78; (mu) a((lambda) ) first decreases with (lambda) till (lambda) approximately equals 0.61 micrometers and then gradually increases with (lambda) . In the Rdink experiments, it has been found that when the port of the integrating sphere is lifted above the liquid surface, the dependence of the measured intensity with the height H can be well-fitted into an exponential relation for H <EQ 3 cm and Lorentzian relation for H <EQ 10 cm, so Rdink can be accurately derived by a simple extrapolation over a few measured points to H equals 0. Monte-Carlo simulation is applied to analyze the results.

Cr,Tm,Ho: YAG laser amplifier

Among other applications, Q-switched, single-mode and high energy Ho:YAG lasers are useful tools in temperature-jump experiments designed for biophysical research. But if pulse energies of 200 J at one microsecond pulse widths or shorter are to be generated in a single oscillator, the damage threshold of the optical coatings is likely to be exceeded. Experiments with an oscillator/amplifier system show that the saturation fluence of Cr,Tm,Ho:YAG amounts to 2.3 J/cm^2 and that a small signal gain coefficient of 0.15 1/cm can be obtained at moderate pump densities. This suggests the use of extracavity amplification if high power holmium pulses are required.

Simple model for the effectiveness of PDT with pulsed-laser sources

Irradiation with pulsed lasers can change mechanisms and efficacy of photodynamic therapy (PDT) depending on the laser pulse parameters. Since most photosensitizers have a relatively high triplet quantum yield and triplet lifetimes of tens of microseconds, even moderate power densities below 100 kW/cm2 can lead to a saturation of the singlet oxygen production, thereby reducing the PDT effect. A simple quantitative model is developed to estimate this effect. According to this, for laser pulses not longer than the triplet lifetime, the PDT efficacy depends on the product of single pulse energy, irradiation wavelength, and the extinction coefficient of the photosensitizer. Peak irradiance and pulse width have minor influence on the efficacy of pulsed irradiation, which decreases as the triplet quantum yield reaches one. The model is supported by in vitro experiments with Photosan 3 and in vivo and in vitro experiments with Photosan 3, Photofrin, aluminum sulphonated phthalocyanine (AlSPc) and benzoporphyrin derivative monoacid ring A (BPD-MA) reported in the literature.

Possibility of measuring thermal protein denaturation by an optoacoustic method

The coupling of proteins to a chromophore allows us, in principle, to achieve a high temperature for a short time in a highly confined microvolume. On the one hand this can be used for the study of very rapid thermal denaturation, which may happen in microseconds. On the other hand it is possible to produce very precise damage to special cellular and subcellular structures by heating a microscopic volume. Preliminary optoacoustic experiments were conducted to explore the possibility of detecting denaturation of proteins in such conjugates by their volume change, which is associated with the change of the tertiary structure. As expected, the melanin granules proved to be efficient photon-to-heat converters. Nevertheless, the volume change due to protein denaturation may be too small compared to the thermal expansion of water to allow quantitative evaluations.