Martin Čapek

Analysis and three-dimensional visualization of collagen in artificial scaffolds using nonlinear microscopy techniques

Extracellularly distributed collagen and chondrocytes seeded in gelatine and poly-ɛ-caprolactone scaffolds are visualized by two-photon excitation microscopy (TPEM) and second-harmonic generation (SHG) imaging in both forward and backward nondescanned modes. Joint application of TPEM and SHG imaging in combination with stereological measurements of collagen enables us not only to take high-resolution 3-D images, but also to quantitatively analyze the collagen volume and a spatial arrangement of cell-collagen-scaffold systems, which was previously impossible. This novel approach represents a powerful tool for the analysis of collagen-containing scaffolds with applications in cartilage tissue engineering.

Volume reconstruction of large tissue specimens from serial physical sections using confocal microscopy and correction of cutting deformations by elastic registration

A set of methods leading to volume reconstruction of biological specimens larger than the field of view of a confocal laser scanning microscope (CLSM) is presented. Large tissue specimens are cut into thin physical slices and volume data sets are captured from all studied physical slices by CLSM. Overlapping spatial tiles of the same physical slice are stitched in horizontal direction. Image volumes of successive physical slices are linked in axial direction by applying an elastic registration algorithm to compensate for deformations because of cutting the specimen. We present a method enabling us to keep true object morphology using a priori information about the shape and size of the specimen, available from images of the cutting planes captured by a USB light microscope immediately before cutting the specimen by a microtome. The errors introduced by elastic registration are evaluated using a stereological point counting method and the Procrustes distance. Finally, the images are enhanced to compensate for the effect of the light attenuation with depth and visualized by a hardware accelerated volume rendering. Algorithmic steps of the reconstruction, namely elastic registration, object morphology preservation, image enhancement, and volume visualization, are implemented in a new Rapid3D software package. Because confocal microscopes get more and more frequently used in scientific laboratories, the described volume reconstruction may become an easy‐to‐apply tool to study large biological objects, tissues, and organs in histology, embryology, evolution biology, and developmental biology. In this work, we demonstrate the reconstruction using a postcranial part of a 17‐day‐old laboratory Wistar rat embryo. Microsc. Res. Tech., 2009.

Comparison of different tissue clearing methods and 3D imaging techniques for visualization of GFP-expressing mouse embryos and embryonic hearts

Our goal was to find an optimal tissue clearing protocol for whole-mount imaging of embryonic and adult hearts and whole embryos of transgenic mice that would preserve green fluorescent protein GFP fluorescence and permit comparison of different currently available 3D imaging modalities. We tested various published organic solvent- or water-based clearing protocols intended to preserve GFP fluorescence in central nervous system: tetrahydrofuran dehydration and dibenzylether protocol (DBE), SCALE, CLARITY, and CUBIC and evaluated their ability to render hearts and whole embryos transparent. DBE clearing protocol did not preserve GFP fluorescence; in addition, DBE caused considerable tissue-shrinking artifacts compared to the gold standard BABB protocol. The CLARITY method considerably improved tissue transparency at later stages, but also decreased GFP fluorescence intensity. The SCALE clearing resulted in sufficient tissue transparency up to ED12.5; at later stages the useful depth of imaging was limited by tissue light scattering. The best method for the cardiac specimens proved to be the CUBIC protocol, which preserved GFP fluorescence well, and cleared the specimens sufficiently even at the adult stages. In addition, CUBIC decolorized the blood and myocardium by removing tissue iron. Good 3D renderings of whole fetal hearts and embryos were obtained with optical projection tomography and selective plane illumination microscopy, although at resolutions lower than with a confocal microscope. Comparison of five tissue clearing protocols and three imaging methods for study of GFP mouse embryos and hearts shows that the optimal method depends on stage and level of detail required.

A perivascular system releasing sirolimus prevented intimal hyperplasia in a rabbit model in a medium-term study

The main complication of aortocoronary reconstruction with vein grafts is restenosis in the course of time. The aim was to assess the effect of a periadventitial polyester mesh releasing sirolimus on intimal hyperplasia of autologous grafts. We implanted v. jugularis ext. into a. carotis communis in rabbits. The vein graft was either intact, or was wrapped with a pure polyester mesh, or with a sirolimus-releasing mesh. Three and six weeks after surgery, the veins were subjected to standard histological staining and the thicknesses of the tunica intima, the media and the intima-media complex were measured. Wrapping the vein with a mesh releasing sirolimus or with a pure mesh decreased the thickness of the intima in comparison with a vein graft by 73 ± 11% or 73 ± 8% after 3 weeks, and by 73 ± 9% or 59 ± 12% after 6 weeks, respectively. Sirolimus-releasing meshes reduced the thickness of the media by 65 ± 9% and 20 ± 12% after 3 and 6 weeks. The thickness of the intima-media complex in grafts with sirolimus-releasing meshes decreased by 60 ± 6% and 30 ± 13% in comparison with pure PES meshes, after 3 and 6 weeks, respectively. A periadventitial polyester mesh releasing sirolimus has the potential to become an effective device in preventing vein graft restenosis.

Testate amoebae examined by confocal and two-photon microscopy: implications for taxonomy and ecophysiology.

Testate amoebae (TA) are a group of free-living protozoa, important in ecology and paleoecology. Testate amoebae taxonomy is mainly based on the morphological features of the shell, as examined by means of light microscopy or (environmental) scanning electron microscopy (SEM/ESEM). We explored the potential applications of confocal laser scanning microscopy (CLSM), two photon excitation microscopy (TPEM), phase contrast, differential interference contrast (DIC Nomarski), and polarization microscopy to visualize TA shells and inner structures of living cells, which is not possible by SEM or environmental SEM. Images captured by CLSM and TPEM were utilized to create three-dimensional (3D) visualizations and to evaluate biovolume inside the shell by stereological methods, to assess the function of TA in ecosystems. This approach broadens the understanding of TA cell and shell morphology, and inner structures including organelles and endosymbionts, with potential implications in taxonomy and ecophysiology.

Compensation of inhomogeneous fluorescence signal distribution in 2D images acquired by confocal microscopy

In images acquired by confocal laser scanning microscopy (CLSM), regions corresponding to the same concentration of fluorophores in the specimen should be mapped to the same grayscale levels. However, in practice, due to multiple distortion effects, CLSM images of even homogeneous specimen regions suffer from irregular brightness variations, e.g., darkening of image edges and lightening of the center. The effects are yet more pronounced in images of real biological specimens. A spatially varying grayscale map complicates image postprocessing, e.g., in alignment of overlapping regions of two images and in 3D reconstructions, since measures of similarity usually assume a spatially independent grayscale map. We present a fast correction method based on estimating a spatially variable illumination gain, and multiplying acquired CLSM images by the inverse of the estimated gain. The method does not require any special calibration of reference images since the gain estimate is extracted from the CLSM image being corrected itself. The proposed approach exploits two types of morphological filters: the median filter and the upper Lipschitz cover. The presented correction method, tested on images of both artificial (homogeneous fluorescent layer) and real biological specimens, namely sections of a rat embryo and a rat brain, proved to be very fast and yielded a significant visual improvement. Microsc. Res. Tech., 2011.

Compensation of the contrast and brightness attenuation with depth in confocal microscopy

A confocal laser scanning microscope (CLSM) captures images from a biological specimen in different depths and provides one with a stack of precisely registered fluorescent images. However, image intensities suffer from light loss distortions showing contrast and brightness degradation of images with depth. This effect causes problems in subsequent analysis and segmentation of biological objects. Therefore, we developed a post-processing method to correct this problem. It is a two step algorithm. In the first step the algorithm determines a standard histogram of the stack of images and in the second step histograms of individual images are warped according to this standard histogram. The algorithm results in more stable brightness and contrast of images through the stack.

Artifact-free 3D Reconstruction for Optical Projection Tomography.

Optical Projection Tomography (OPT) is a recently developed implementation of computed tomography (CT) techniques at optical frequencies. A series of 2D optical projections through a sample are generated at varying orientations, from which the 3D structure of the sample can be computationally reconstructed. OPT is especially suitable for samples from about 0.5 mm to 15 mm in size, which fills an important “imaging gap” between techniques such as confocal microscopy (useful for smaller samples) and large-sample methods such as fluorescence molecular tomography (FMT), x-ray CT or microscopic magnetic resonance imaging (μΜRI). OPT can function in both fluorescence and transmission modes.

Ecology of Testate Amoebae in the Komořany Ponds in the Vltava Basin

Testate amoeba (TA) assemblages were collected in 2005 from four ponds in Komořany (Prague, Czech Republic). An analysis of seasonal taxonomic variability of TA populations and its correlation with the limnological characteristics of the area (temperature, pH, total organic carbon, nitrogen, phosphorus, heavy metals, etc.) was performed. The predominant genera were Difflugia, Arcella, and Centropyxis. The most significant changes in the TA community occurred between March and July. Arcella genus dominated in March and April; in May, Arcella and Centropyxis genera were present in the same amount; in June, Arcella genus disappeared, and Difflugia genus started to dominate the community. A multivariate redundancy analysis showed statistically significant correlations between the environmental parameters and the composition of the TA community. The results indicate a negative correlation between TA quantities and Ni, Cd, PAH, Mn, As, and Pb. TA were also affected by concentrations of NH4+, NO3−, and P, as well as by temperature variations. The observed correlations between the species composition and environmental parameters can be used in paleoecological interpretations of fossil TA communities. Our results also prove the suitability of TA as water quality indicators in urban areas.

Nonrigid registration of CLSM images of physical sections with discontinuous deformations.

When biological specimens are cut into physical sections for three-dimensional (3D) imaging by confocal laser scanning microscopy, the slices may get distorted or ruptured. For subsequent 3D reconstruction, images from different physical sections need to be spatially aligned by optimization of a function composed of a data fidelity term evaluating similarity between the reference and target images, and a regularization term enforcing transformation smoothness. A regularization term evaluating the total variation (TV), which enables the registration algorithm to account for discontinuities in slice deformation (ruptures), while enforcing smoothness on continuously deformed regions, was proposed previously. The function with TV regularization was optimized using a graph-cut (GC) based iterative solution. However, GC may generate visible registration artifacts, which impair the 3D reconstruction. We present an alternative, multilabel TV optimization algorithm, which in the examined samples prevents the artifacts produced by GC. The algorithm is slower than GC but can be sped up several times when implemented in a multiprocessor computing environment. For image pairs with uneven brightness distribution, we introduce a reformulation of the TV-based registration, in which intensity-based data terms are replaced by comparison of salient features in the reference and target images quantified by local image entropies.