Microorganisms can be found everywhere e.g. in food both as useful ingredients or harmful contaminations causing
food spoilage. Therefore, a fast and easy to handle analysis method is needed to detect bacteria in different kinds of
samples like meat, juice or air to decide if the sample is contaminated by harmful microorganisms. Conventional
identification methods in microbiology require always cultivation and therefore are time consuming.
In this contribution we present an analysis approach to identify fluorescence stained bacteria on strain level by means of
Raman spectroscopy. The stained bacteria are highlighted and can be localized easier against a complex sample
environment e.g. in food. The use of Raman spectroscopy in combination with chemometrical methods allows the
identification of single bacteria within minutes.
Irradiation of 15 nm gold particles with nano- and picosecond laser pulses can create locally temperatures beyond the critical point of water. Due to the short heating times the temperature is localised to the vicinity of the particles. Under irradiation with nano- and picosecond pulses an inactivation of the enzymes alkaline phosphatase and chymotrypsin which were bound to the surface of the particles was observed. As expected by strong temperature gradient caused by
the short irradiation time the protein inactivation is localized within a few tens of nanometers when picosecond pulses were used. This was shown by irradiating gold protein conjugates in which the protein was bound via two anitbodies to the particle.
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 Mie's 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.
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 Mie's 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.
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 2.3J/cm2 and that a small signal gain coefficient of 0.15/cm can be obtained at moderate pump densities. This suggests the use of extracavity ampli cation if high power holmium pulses are required.
Laser thermokeratoplasty (LTK) is a minimally invasive method to correct hyperopia and astigmatism. A cw mid-IR laser diode emitting at wavelengths around 1.86 micrometers was used to perform LTK on a first clinical trial. The coagulations were applied to the cornea by means of a specially designed focusing handpiece which was introduced into a corneal application mask fixed by a suction ring. Coagulation patterns consisting of 8 spots per ring were performed with a laser power between 100 - 150 mW and an irradiation time of 10 seconds both on single and on double rings. Significant refractive changes up to 19 D could initially be achieved followed by a strong regression within the first month. Three months post LTK, refractive changes achieved with the single and double ring have stabilized, yielding 1.2 and 1.8 D on the average, respectively. The method reveals only little adverse effects limited to the first days post-op. Force measurements were performed on corneal stripes, which were submerged for 10 s into an oil bath of constant temperature in order to investigate the absolute temperatures required for corneal collagen contraction. Only temperatures exceeding 90 degree(s)C induced a significant force. Analyzing the clinically used LTK parameters by temperature calculations revealed that only a small part of the heated stromal volume experienced sufficient high temperatures to induce significant collagen shrinkage.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.