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.

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.

Two-photon induced collagen cross-linking in bioartificial cardiac tissue.

Cardiac tissue engineering is a promising strategy for regenerative therapies to overcome the shortage of donor organs for transplantation. Besides contractile function, the stiffness of tissue engineered constructs is crucial to generate transplantable tissue surrogates with sufficient mechanical stability to withstand the high pressure present in the heart. Although several collagen cross-linking techniques have proven to be efficient in stabilizing biomaterials, they cannot be applied to cardiac tissue engineering, as cell death occurs in the treated area. Here, we present a novel method using femtosecond (fs) laser pulses to increase the stiffness of collagen-based tissue constructs without impairing cell viability. Raster scanning of the fs laser beam over riboflavin-treated tissue induced collagen cross-linking by two-photon photosensitized singlet oxygen production. One day post-irradiation, stress-strain measurements revealed increased tissue stiffness by around 40% being dependent on the fibroblast content in the tissue. At the same time, cells remained viable and fully functional as demonstrated by fluorescence imaging of cardiomyocyte mitochondrial activity and preservation of active contraction force. Our results indicate that two-photon induced collagen cross-linking has great potential for studying and improving artificially engineered tissue for regenerative therapies.

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.

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.

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.