Marko Heidrich

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.

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.

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.

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.

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.

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.

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.

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

New optical techniques have the potential to fill the gap between radiological and microscopic approaches to assess the lungs internal structure. Since its quantitative assessment requires unbiased sampling and measurement principles, imaging of the whole lung with sufficient resolution for visualizing details is important. To address this request, we applied scanning laser optical tomography (SLOT) for the three dimensional imaging of mouse lung ex vivo. SLOT is a highly efficient flourescence and transmission microscopy technique allowing for 3D imaging of specimen of sizes up to several millimeters. Previously fixed lung lobes and whole lungs were optically cleared and subsequently imaged with SLOT while making use of intrinsic contrast mechanisms like absorption and autofluorescence. Imaging of airways, blood vessels and parenchyma is demonstrated. Volumetric SLOT datasets of the lungs internal structure can be analyzed in any preferred planar orientation. Moreover, the sample preparation preserves microscopic structure of the lung and allows for subsequent correlative histologic studies. In summary, SLOT is a useful technique to visualize and survey the internal structure of mouse lung at different scales and with various contrast mechanisms. Potential applications of SLOT in lung research are e.g. quantitative phenotype analysis of mouse models of human lung disease in combination with stereological methods.