Raoul-Amadeus Lorbeer

Highly efficient 3D fluorescence microscopy with a scanning laser optical tomograph

Optical Projection Tomography (OPT) proved to be useful for the three-dimensional tracking of fluorescence signals in biological model organisms with sizes up to several millimeters. This tomographic technique detects absorption as well as fluorescence to create multimodal three-dimensional data. While the absorption of a specimen is detected very fast usually less than 0.1% of the fluorescence photons are collected. The low efficiency can result in radiation dose dependent artifacts such as photobleaching and phototoxicity. To minimize these effects as well as artifacts introduced due to the use of a CCD- or CMOS- camera-chip, we constructed a Scanning Laser Optical Tomograph (SLOT). Compared to conventional fluorescence OPT our first SLOT enhanced the photon collection efficiency a hundredfold.

3D imaging of biofilms on implants by detection of scattered light with a scanning laser optical tomograph

Biofilms – communities of microorganisms attached to surfaces – are a constant threat for long-term success in modern implantology. The application of laser scanning microscopy (LSM) has increased the knowledge about microscopic properties of biofilms, whereas a 3D imaging technique for the large scale visualization of bacterial growth and migration on curved and non-transparent surfaces is not realized so far. Towards this goal, we built a scanning laser optical tomography (SLOT) setup detecting scattered laser light to image biofilm on dental implant surfaces. SLOT enables the visualization of living biofilms in 3D by detecting the wavelength-dependent absorption of non-fluorescent stains like e.g. reduced triphenyltetrazolium chloride (TTC) accumulated within metabolically active bacterial cells. Thus, the presented system allows the large scale investigation of vital biofilm structure and in vitro development on cylindrical and non-transparent objects without the need for fluorescent vital staining. We suggest SLOT to be a valuable tool for the structural and volumetric investigation of biofilm formation on implants with sizes up to several millimeters.

Scanning Laser Optical Tomography Resolves Structural Plasticity during Regeneration in an Insect Brain

Background Optical Projection Tomography (OPT) is a microscopic technique that generates three dimensional images from whole mount samples the size of which exceeds the maximum focal depth of confocal laser scanning microscopes. As an advancement of conventional emission-OPT, Scanning Laser Optical Tomography (SLOTy) allows simultaneous detection of fluorescence and absorbance with high sensitivity. In the present study, we employ SLOTy in a paradigm of brain plasticity in an insect model system. Methodology We visualize and quantify volumetric changes in sensory information procession centers in the adult locust, Locusta migratoria. Olfactory receptor neurons, which project from the antenna into the brain, are axotomized by crushing the antennal nerve or ablating the entire antenna. We follow the resulting degeneration and regeneration in the olfactory centers (antennal lobes and mushroom bodies) by measuring their size in reconstructed SLOTy images with respect to the untreated control side. Within three weeks post treatment antennal lobes with ablated antennae lose as much as 60% of their initial volume. In contrast, antennal lobes with crushed antennal nerves initially shrink as well, but regain size back to normal within three weeks. The combined application of transmission-and fluorescence projections of Neurobiotin labeled axotomized fibers confirms that recovery of normal size is restored by regenerated afferents. Remarkably, SLOTy images reveal that degeneration of olfactory receptor axons has a trans-synaptic effect on second order brain centers and leads to size reduction of the mushroom body calyx. Conclusions This study demonstrates that SLOTy is a suitable method for rapid screening of volumetric plasticity in insect brains and suggests its application also to vertebrate preparations.

A combined method for correlative 3D imaging of biological samples from macro to nano scale.

Correlative analysis requires examination of a specimen from macro to nano scale as well as applicability of analytical methods ranging from morphological to molecular. Accomplishing this with one and the same sample is laborious at best, due to deformation and biodegradation during measurements or intermediary preparation steps. Furthermore, data alignment using differing imaging techniques turns out to be a complex task, which considerably complicates the interconnection of results. We present correlative imaging of the accessory rat lung lobe by combining a modified Scanning Laser Optical Tomography (SLOT) setup with a specially developed sample preparation method (CRISTAL). CRISTAL is a resin-based embedding method that optically clears the specimen while allowing sectioning and preventing degradation. We applied and correlated SLOT with Multi Photon Microscopy, histological and immunofluorescence analysis as well as Transmission Electron Microscopy, all in the same sample. Thus, combining CRISTAL with SLOT enables the correlative utilization of a vast variety of imaging techniques.

Experimental verification of high energy laser-generated impulse for remote laser control of space debris

Walking along a beach one may notice debris being washed ashore from the vast oceans. Then, turning your head up at night you even might noticed a shooting star or a bright spot passing by. Chances are, that you witnessed space debris, endangering future space flight in lower earth orbit. If it was possible to turn cm-sized debris into shooting stars the problem might be averted. Unfortunately, these fragments counting in the 100 thousands are not controllable. To possibly regain control we demonstrate how to exert forces on a free falling debris object from a distance by ablating material with a high energy ns-laser-system. Thrust effects did scale as expected from simulations and led to speed gains above 0.3 m/s per laser pulse in an evacuated micro-gravity environment.

Correction of image artifacts caused by refractive index gradients in scanning laser optical tomography

We present a technique for correcting image artifacts caused by refractive index distributions in Scanning Laser Optical Tomography (SLOT) and Optical Projection Tomography (OPT). Projection images can be distorted due to the presence of a refractive index distribution around the sample. We consider the special case of a refractive index distribution given by a capillary around a sample. The particular application we are interested in is in vitro imaging of cell spheroids in a glass capillary. Numerical simulations and experimental results are used to illustrate the connection between the Radon transform and the refracted projection. Thereupon we will describe a technique that transforms refracted projections to parallel ray Radon projections and thus allows artifact free reconstruction within the sample volume.

Applying optical Fourier filtering to standard optical projection tomography

Light microscopy is one of the major tools in modern biology. The steady development of new microscopic techniques leads to an correspondent improvement of biological methods. To expand the catalog of biological experiments, we investigate the possibilities of optical projection tomography (OPT). This technique is based on the already established X-Ray computed tomography. In contrast to most other three-dimensional microscopy techniques it is able to create three dimensional data sets of the specimens natural absorption, staining and fluorescence. Unfortunately, these advantages are opposed by a low resolution, reconstruction artifacts, and a relatively big loss of fluorescence light. We reduced the disadvantage in resolution by applying physical filters in the Fourier plane of the image path, which is not possible in X-Ray imaging yet.

Thrust noise minimization in long-term laser ablation of propellant material in the nanosecond and picosecond regime

Abstract. The avoidance of any moving parts in a microthruster exhibits a great potential for low-noise thrust generation in the micronewton range. This is required, e.g., for scientific missions that need attitude and orbit control systems with exquisite precision. Laser ablation propulsion offers the opportunity of permanent inertia-free, electro-optical delivery of laser energy to access the propellant entirely without moving it. New propellant is accessed by ablating the previous surface in layers, essentially damaging the surface with a laser over and over again. The resulting surface properties for different fluences and scanning patterns were investigated for multiple layers of aluminum, copper, and gold. The pulse-length-specific issues of various ablation mechanisms such as vaporization, spallation, and phase explosion are accounted for by the use of a 10-ps laser system and a 500-ps laser system. We show that the surface roughness produced with 500-ps laser pulses is approximately twice the surface roughness generated by using 10-ps laser pulses. Furthermore, with 500-ps pulses, the surface roughness shows low dependency on the fluence for carefully chosen scanning parameters. Therefore, we conclude that laser pulse duration differences in the picosecond and nanosecond regimes will not necessarily alter surface roughness properties.

Method for 3D airway topology extraction.

In lungs the number of conducting airway generations as well as bifurcation patterns varies across species and shows specific characteristics relating to illnesses or gene variations. A method to characterize the topology of the mouse airway tree using scanning laser optical tomography (SLOT) tomograms is presented in this paper. It is used to test discrimination between two types of mice based on detected differences in their conducting airway pattern. Based on segmentations of the airways in these tomograms, the main spanning tree of the volume skeleton is computed. The resulting graph structure is used to distinguish between wild type and surfactant protein (SP-D) deficient knock-out mice.

Three dimensional numerical simulation of complex optical systems using the coherent transfer function

To be able to understand and improve various optical systems including fs-laser lightsources, we developed a numerical simulation based on the coherent transfer function (CTF). Our work created a simulation tool, which is able to simulate all three dimensions and time while accounting for coherent light, short pulses, aberrations, chirp, pulse delay, numerical aperture, the intensity distribution and the polarization of the incident light. We explain the theoretical approach and aspects necessary for the simulations. Then we show the capabilities of our tool in simulation aberrations, 4Pi point spread functions, two objective fs-pulse interaction and multi objective confocal fluorescence microscopy. Keywords: numerical, femtosecond, focus, coherent transfer function (CTF), optical transfer function (OTF), fourier optics, aberration, polarization