Synthetic dispersion interferometry for relative atmospheric pressure sensing.

We present a modified version of the two-arm, two-color, single second harmonic generation heterodyne dispersion interferometer, as introduced by Irby et. al. [Rev. Sci. Instrum.70, 699 (1999) 10.1063/1.1149489]. The amount of optical elements is reduced and digital in-phase and quadrature demodulation is used to retrieve the phase shift from a single photodetector signal. The intrinsic system noise and drift for this device are analyzed by measuring the Allan deviation. We investigate the use of this device for relative atmospheric pressure measurement. Relative pressure measurements are performed in a pressure chamber and referenced against a piezoresistive pressure transceiver to demonstrate the concept. It was found that the deviation was less than 150 Pa and an error estimation has been derived.

Correlating 3D morphology with molecular pathology: fibrotic remodelling in human lung biopsies.

Assessing alterations of the parenchymal architecture is essential in understanding fibrosing interstitial lung diseases. Here, we present a novel method to visualise fibrotic remodelling in human lungs and correlate morphological three-dimensional (3D) data with gene and protein expression in the very same sample. The key to our approach is a novel embedding resin that clears samples to full optical transparency and simultaneously allows 3D laser tomography and preparation of sections for histology, immunohistochemistry and RNA isolation. Correlating 3D laser tomography with molecular diagnostic techniques enables new insights into lung diseases. This approach has great potential to become an essential tool in pulmonary research.

Numerical far field simulations with the fast Fourier transformation and Fourier space interpolation.

As more complicated microscope systems are engineered, the amount of effects taken into account rises steadily. In this context we experienced the need for a simulation approach, that will deliver the intensity distribution in space and time for scanning laser microscopes. To achieve this goal, the frequency space representation of microscope objectives was used and adapted to determine their solution of the electromagnetic wave equation. We describe the steps necessary to efficiently implement an approach to simulate multidimensional solutions of the wave equation. This includes the connection between the back focal plane and the Fourier space representation as well as a proper interpolation method for the latter. The error-potential of our least erroneous interpolation, the power of hann (POH) interpolation, is compared to other common interpolation methods. Finally we demonstrate the current potential of the approach by simulating an "expanding" optical vortex focus.

Single shot telecentricity measurement by Fourier space grid separation.

The experimental documentation of the properties of an optical system represents a particular challenge. Besides the measurement of focal quality and field distortions, telecentric systems have to yield a parallel beam propagation direction. In this paper we propose a method to test, document and optimize the telecentricity of a laser scanning system by scanning two crossed polka dot beam splitters at once. By separating both beam splitters in Fourier space we were able to detect tilting angles below 2 · 10(-3) rad for four different laser wavelengths within the same optical system. By this we determined the optimum system parameters for our scanning laser optical tomography (SLOT) setup.

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.

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.

Power Spectral Density Evaluation of Laser Milled Surfaces.

Ablating surfaces with a pulsed laser system in milling processes often leads to surface changes depending on the milling depth. Especially if a constant surface roughness and evenness is essential to the process, structural degradation may advance until the process fails. The process investigated is the generation of precise thrust by laser ablation. Here, it is essential to predict or rather control the evolution of the surfaces roughness. Laser ablative milling with a short pulse laser system in vacuum (≈1 Pa) were performed over depths of several 10 µm documenting the evolution of surface roughness and unevenness with a white light interference microscope. Power spectral density analysis of the generated surface data reveals a strong influence of the crystalline structure of the solid. Furthermore, it was possible to demonstrate that this effect could be suppressed for gold.

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.

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.

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.

Imaging of the mouse lung with scanning laser optical tomography (SLOT).

The current study focuses on the use of scanning laser optical tomography (SLOT) in imaging of the mouse lung ex vivo. SLOT is a highly efficient fluorescence microscopy technique allowing rapid scanning of samples of a size of several millimeters, thus enabling volumetric visualization by using intrinsic contrast mechanisms of previously fixed lung lobes. Here, we demonstrate the imaging of airways, blood vessels, and parenchyma from whole, optically cleared mouse lung lobes with a resolution down to the level of single alveoli using absorption and autofluorescence scan modes. The internal structure of the lung can then be analyzed nondestructively and quantitatively in three-dimensional datasets in any preferred planar orientation. Moreover, the procedure preserves the microscopic structure of the lung and allows for subsequent correlative histologic studies. In summary, the current study has shown that SLOT is a valuable technique to study the internal structure of the mouse lung.

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.