BACKGROUND: In the field of hearing research a variety of imaging techniques are available to study molecular and cellular structures of the cochlea. Most of them are based on decalcifying, embedding, and cutting of the cochlea. By means of scanning laser optical tomography (SLOT), the complete cochlea can be visualized without cutting. The Cav1.3-/- mice have already been extensively characterized and show structural changes in the inner ear. Therefore, they were used in this study as a model to investigate whether SLOT can detect structural differences in the murine cochlea. MATERIALS AND METHODS: Whole undissected cochleae from Cav1.3-/- and wild-type mice of various postnatal stages were immunostained and analyzed by SLOT. The results were compared to cochlea preparations that were immunostained and analyzed by fluorescence microscopy. In addition, cochlea preparations were stained with osmium tetraoxide. RESULTS: Visualization by SLOT showed that the staining of nerve fibers at P27 in Cav1.3-/- mice was almost absent compared to wild-type mice and earlier timepoints (P9). The analysis of cochlea preparations confirmed a reduction of the radial nerve fibers. In addition, a significantly reduced number of ribbon synapses per inner hair cell (IHC) at P20 and P27 in the apical part of the cochlea of Cav1.3-/- mice was detected. CONCLUSION: The visualization of whole non-dissected cochleae by SLOT is a suitable tool for the analysis of gross phenotypic changes, as demonstrated by means of the Cav1.3-/- mouse model. For the analysis of finer structures of the cochlea, however, further methods must be used.
Cochlea , Hair Cells, Auditory, Inner/ultrastructure , Tomography, Optical , Animals , Disease Models, Animal , Mice , Synapses , Tomography, Optical/methods
BACKGROUND: In the field of hearing research a variety of imaging techniques are available to study molecular and cellular structures of the cochlea. Most of them are based on decalcifying, embedding, and cutting of the cochlea. By means of scanning laser optical tomography (SLOT), the complete cochlea can be visualized without cutting. The Cav1.3-/- mice have already been extensively characterized and show structural changes in the inner ear. Therefore, they were used in this study as a model to investigate whether SLOT can detect structural differences in the murine cochlea. MATERIALS AND METHODS: Whole undissected cochleae from Cav1.3-/- and wildtype mice of various postnatal stages were immunostained and analyzed by SLOT. The results were compared to cochlea preparations that were immunostained and analyzed by fluorescence microscopy. In addition, cochlea preparations were stained with osmium tetraoxide. RESULTS: Visualization by SLOT showed that the staining of nerve fibers at P27 in Cav1.3-/- mice was almost absent compared to wildtype mice and earlier timepoints (P9). The analysis of cochlea preparations confirmed a reduction of the radial nerve fibers. In addition, a significantly reduced number of ribbon synapses per inner hair cell (IHC) at P20 and P27 in the apical part of the cochlea of Cav1.3-/- mice was detected. CONCLUSION: The visualization of whole non-dissected cochleae by SLOT is a suitable tool for the analysis of gross phenotypic changes, as demonstrated by means of the Cav1.3-/- mouse model. For the analysis of finer structures of the cochlea, however, further methods must be used.
Hair Cells, Auditory, Inner , Tomography, Optical , Animals , Cochlea , Disease Models, Animal , Hair Cells, Auditory, Inner/ultrastructure , Mice , Synapses , Tomography, Optical/methods
Optical stimulation of the cochlea with laser light has been suggested as an alternative to conventional treatment of sensorineural hearing loss with cochlear implants. The underlying mechanisms are controversially discussed: The stimulation can either be based on a direct excitation of neurons, or it is a result of an optoacoustic pressure wave acting on the basilar membrane. Animal studies comparing the intra-cochlear optical stimulation of hearing and deafened guinea pigs have indicated that the stimulation requires intact hair cells. Therefore, optoacoustic stimulation seems to be the underlying mechanism. The present study investigates optoacoustic characteristics using pulsed laser stimulation for in vivo experiments on hearing guinea pigs and pressure measurements in water. As a result, in vivo as well as pressure measurements showed corresponding signal shapes. The amplitude of the signal for both measurements depended on the absorption coefficient and on the maximum of the first time-derivative of laser pulse power (velocity of heat deposition). In conclusion, the pressure measurements directly demonstrated that laser light generates acoustic waves, with amplitudes suitable for stimulating the (partially) intact cochlea. These findings corroborate optoacoustic as the basic mechanism of optical intra-cochlear stimulation.
Acoustic Stimulation/methods , Cochlea/physiology , Lasers , Action Potentials , Animals , Female , Guinea Pigs , Hair Cells, Auditory , Male
Nonlinear microscopy is a non-invasive imaging technique which allows a visualization of biological tissue with high signal contrast due to spectral separation combined with high resolution. In addition to two-photon fluorescence and second harmonic signals also four-wave mixing signals can be used for imaging ocular structures. This review article presents the physical principles of different contrast mechanisms. Exemplary experimental results based on various nonlinear signals are shown, opportunities of this technology are discussed and the prospect of translating this imaging technique into a clinical application is addressed.
Eye Diseases/pathology , Image Enhancement/instrumentation , Microscopy, Confocal/instrumentation , Microscopy, Fluorescence, Multiphoton/instrumentation , Ophthalmoscopes , Equipment Design , Equipment Failure Analysis , Humans , Image Enhancement/methods , Microscopy, Confocal/methods , Microscopy, Fluorescence, Multiphoton/methods , Nonlinear Dynamics , Technology Assessment, Biomedical
Nanoparticle laser interactions are in widespread use in cell manipulation. In particular, molecular medicine needs techniques for the directed delivery of molecules into mammalian cells. Proteins are the final mediator of most cellular cascades. However, despite several methodical approaches, the efficient delivery of proteins to cells remains challenging. This paper presents a new protein transfection technique via laser scanning of cells previously incubated with gold nanoparticles. The laser-induced plasmonic effects on the gold nanoparticles cause a transient permeabilization of the cellular membrane, allowing proteins to enter the cell. Applying this technique, it was possible to deliver green fluorescent protein into mammalian cells with an efficiency of 43%, maintaining a high level of cell viability. Furthermore, a functional delivery of Caspase 3, an apoptosis mediating protein, was demonstrated and evaluated in several cellular assays. Compared to conventional protein transfection techniques such as microinjection, the methodical approach presented here enables high-throughput transfection of about 10 000 cells per second. Moreover, a well-defined point in time of delivery is guaranteed by gold nanoparticle mediated laser transfection, allowing the detailed temporal analysis of cellular pathways and protein trafficking.
Caspase 3/metabolism , Caspase 3/pharmacology , Cytological Techniques , Gold/chemistry , Green Fluorescent Proteins/metabolism , Lasers , Metal Nanoparticles/chemistry , Animals , Apoptosis/drug effects , Cell Line, Tumor , Cell Membrane Permeability/drug effects , Cell Survival/drug effects , Dogs , Green Fluorescent Proteins/pharmacology , Metal Nanoparticles/toxicity
Animal experiments suggest that pharmacological intervention could possibly enhance cochlear implant performance. One of the key aspects is therefore a drug delivery device for the human inner ear. The objective of this study was to investigate the possibility of using the femtosecond laser for modifying a cochlear implant electrode for the purpose of drug delivery to the cochlea. Using silicone sheets, the best parameters for creating defined channels at calculated diameters were investigated using a femtosecond laser. The results were transferred to a cochlear implant electrode array (Nucleus Contour). The capability of delivering substances through the drilled openings was tested in vitro. By variation of the output of the laser, spot distance, repetition rate, number of cycles and introducing several focus planes, it was possible to drill holes with nearly vertical walls in the silicone sheets. Transferring these data to the cochlear implant electrode resulted in prototypes for drug delivery with various openings along the array. The use of the femtosecond laser allows rapid modification and adaptation of designs to experimental prototypes of cochlear implant electrodes for the purpose of drug delivery to the inner ear.
Cochlea/drug effects , Cochlear Implants , Drug Delivery Systems/instrumentation , Lasers , Electrodes, Implanted , Feasibility Studies , Humans , In Vitro Techniques
BACKGROUND: Ultrashort (femtosecond) laser pulses can generate precise cuts in biological tissue without damaging the surface. The application of femtosecond laser technology at the lens was evaluated with respect to a possible treatment of presbyopia. MATERIALS AND METHODS: Femtosecond laser lentotomy was performed on 150 pig lenses in vitro. Cutting geometry and laser settings were optimized to generate smooth cuts with a minimum of produced gas bubbles. Four rabbit lenses were treated afterwards in vivo and were controlled for 3 months post-treatment. The lenses were then extracted and evaluated. RESULTS: With suitable laser settings, light scattering due to residual gas bubbles could be almost completely avoided in pig lenses. A pulse energy of less than 1.2 microJ and a cutting geometry with spot separations of more than 5 microm are important. The rabbit lenses stayed macroscopically clear for 3 months in vivo. Only the cell structures directly adjacent to the laser focus were cut; structures 5-10 microm away appeared to be intact. No cataract formation occurred during this time. CONCLUSION: Femtosecond laser application allows precise and smooth cuts inside pig and rabbit lenses without damage to adjacent tissue.
Laser Therapy/instrumentation , Lens, Crystalline/pathology , Lens, Crystalline/surgery , Presbyopia/pathology , Presbyopia/surgery , Animals , Equipment Design , Equipment Failure Analysis , Laser Therapy/methods , Swine , Treatment Outcome
BACKGROUND: With the assistance of ultrashort laser pulses (ca. 200 fs pulse duration) it is possible to perform precise incisions inside the corneal stroma with a width of a few microns. The advantage of ultrashort pulses is that the required energy of a few microjoules is more than an order of magnitude lower compared with longer pulse durations, i.e. ps or ns pulses. Therefore, the secondary effects, such as thermal and mechanical damage to the surrounding tissue and the amount of radiation reaching the retina, are reduced. This method of intrastromal photodisruption allows a very well defined deposition of energy within the laser focus inside the corneal stroma, accompanied by minimal collateral damage. METHODS: The possibilities of performing intrastromal cuts using fs-laser pulses at a wavelength of 780 nm and pulse durations of 200 fs were studied using a titanium-sapphire laser system. The treated tissue samples were analysed by light and scanning electron microscopy to determine incision quality, reproducibility and achievable accuracy. The mechanical side effects of fs-photodisruption inside the surrounding tissue were analysed by pressure measurements using pyroelectric transducers. CONCLUSION: The thermal and mechanical side-effects of this method are very low and comparable to the effects during excimer treatment. Therefore an application of ultrashort laser pulses in refractive surgery appears to be a feasible alternative.
Corneal Stroma/surgery , Keratomileusis, Laser In Situ/instrumentation , Animals , Corneal Stroma/pathology , Equipment Design , Microscopy, Electron, Scanning , Swine
