KEYWORDS: Image segmentation, Denoising, Optical flow, Luminescence, Proteins, Wavelets, In vitro testing, 3D image processing, Signal to noise ratio, Image processing algorithms and systems
We present an approach for the computation of single-object velocity statistics in a noisy fluorescence image series. The algorithm is applied to molecular imaging data from an in vitro actin-myosin motility assay. We compare the relative efficiency of wavelet and curvelet transform denoising in terms of noise reduction and object restoration. It is shown that while both algorithms reduce background noise efficiently, curvelet denoising restores the curved edges of actin filaments more reliably. Noncrossing spatiotemporal actin trajectories are unambiguously identified using a novel segmentation scheme that locally combines the information of 2-D and 3-D segmentation. Finally, the optical flow vector field for the image sequence is computed via the 3-D structure tensor and mapped to the segmented trajectories. Using single-trajectory statistics, the global velocity distribution extracted from an image sequence is decomposed into the contributions of individual trajectories. The technique is further used to analyze the distribution of the x and y components of the velocity vectors separately, and it is shown that directed actin motion is found in myosin extracts from single skeletal muscle fibers. The presented approach may prove helpful to identify actin filament subpopulations and to analyze actin-myosin interaction kinetics under biochemical regulation.
KEYWORDS: Image segmentation, Denoising, Molecules, Signal to noise ratio, Image processing, Wavelets, Luminescence, Image processing algorithms and systems, Digital filtering, 3D image processing
We present a multiresolution transform-based method for the extraction of moving filament trajectories from single
molecule motility data. Noise-corrupted fluorescence image series are denoised using the multiscale median transform
and trajectories are detected in the denoised data set. The presented method reduces noise more efficiently than 2D-anisotropic
diffusion and several wavelet based techniques. Fibre trajectories are extracted by segmentation of the
denoised image stacks and non-crossing trajectories are unambiguously identified combining the information of 2D (XY)
and 3D (XYT) segmentation.
The algorithm is applied and evaluated using experimental data sets - image sequences of fluorescently labeled F-actin
molecules and their 2D-trajectories on a myosin coated surface. This so-called 'motility assay' is used to analyse
kinetics, biochemical regulation and pharmacological modulation of these biologically relevant molecules. The presented
method improves signal-to-background discrimination, facilitates filament identification and finally, may contribute to
significantly improve the performance of this assay.
Intrinsic second harmonic generation (SHG) signals can be used to visualize the three-dimensional structure of cardiac and skeletal muscle with high spatial resolution. Fluorescence labeling of complementary sarcomeric proteins, e.g. actin, indicates that the observed SHG signals arise from the myosin filaments.
Recently, the myosin rod domain or LMM - light meromyosin - has been reported to be the dominant source of this SHG signal. However, to date, mostly negative and indirect evidence has been presented to support this assumption.
Here, we show, to our knowledge, the first direct evidences that strong SHG signals can be obtained from synthetic paracrystals. These rod shaped filaments are formed from purified LMM. SDS-PAGE protein analysis confirmed that the LMM crystals lack myosin head domains. Some regions of the LMM paracrystals produce a strong SHG signal whereas others did not.
The SHG signals were recorded with a laser-scanning microscope (Leica SP2). A ps laser tuned to 880 nm was used to excite the sample through an 63x objective of 1.2 NA. In order to visualize the synthetic filaments - in addition to SHG imaging -, the LMM was labeled with the fluorescent marker 5-IAF. We were able to observe filaments of 1 to 50 μm in length and of up to 5 μm in diameter.
In conclusion, we can show that the myosin rod domain (LMM) is a dominant source for intrinsic SHG signals. There seems, however, a signal dependence on the paracrystals' morphology. This dependence is being investigated.
Intrinsic Second Harmonic Generation (SHG) signals obtained from the motor protein myosin are of particular interest for 3D-imaging of living muscle cells. In addition, the new and powerful tool of 4Pi microscopy allows to markedly enhance the optical resolution of microscopy as well as the sensitivity for small objects because of the high peak intensities due to the interference pattern created in the focus. In the present study, we report, to our knowledge for the first time, measurements of intrinsic SHG signals under 4Pi conditions of type A. These measurements on mammalian myofibrilar structures are combined with very high resolution 4Pi fluorescence data obtained from the same preparations. We have chosen myofibrillar preparations of isolated mammalian muscle fibers as they (i) possess a regular repetitive pattern of actin and myosin filaments within sarcomers 2 to 3 μm in length, (ii) consist of single myofibrils of small total diameter of approximately 1 μm and (iii) are ideally suited to study the biomedically important process of force generation via calcium regulated motor protein interactions. Myofibrillar preparations were obtained from murine skeletal and heart muscle by using a combined chemical and mechanical fractionation1 (Both et al. 2004, JBO 9(5):882-892). BODIPY FL phallacidin has been used to fluorescently label the actin filaments.
The experiments were carried out with a Leica SP2 multi photon microscope modified for 4Pi measurements using a Ti:Sa laser tuned to 850-900 nm. SHG as well as fluorescence photons were detected confocally by a counting APD detector. The approach taken our study provides new 3D-data for the analysis and simulation of the important process of excitation-contraction coupling under normal physiological as well as under pathophysiological conditions.
We have recently shown that intrinsic, chromophore free Second Harmonic Generation (SHG) signals can be obtained from myofibrillar structures of mammalian skeletal muscle1,2 (Both et al. 2003, Proc. SPIE 5139: 112-120; Both et al. 2004, JBO 9(5):882-892). Here, we report experiments at the level of single myofibrils (diameters 1 to 2 µm) to characterize the spatial dependency of the hyperpolarizability chi(2) and to generate a map of this tensor in myofibrillar structures. Myofibrils are the smallest functional sub cellular contractile structures of muscle. They are organized in a regular sarcomer pattern with a periodicity of 2 to 3 µm. Single myofibrils were obtained from mammalian skeletal muscle using a combined chemical and mechanical fractionation. The SHG signals were recorded with an inverse laser scanning microscope (Leica SP2). A ps laser source (Ti:Sa laser, Tsunami, Spectra Physics) tuned to 880 nm was used to excite the sample through an objective of high NA (1.2NA, 63x). The laser source was linearly polarized and the axis of polarization could be adjusted in steps of degrees with a half-wave plate. The forward scattered SHG signal was collected with a matching objective placed above the preparation. The SHG signals depend both on polarization and location within the myofibrillar structures. The SHG signals seem to arise from the myosin molecules. In conclusion, SHG imaging allows to monitor the myofibrillar structure with two photon resolution.
We use second harmonic generation (SHG) imaging to study and quantify a strong intrinsic SHG signal in skeletal muscle fiber preparations and single isolated myofibrils. The intrinsic signal follows the striation pattern of the muscle cells and is positioned at the sarcomeric location of the myosin filaments. Interestingly, the signal is enhanced at the region where the myosin heads are located on the myosin filaments. As the intrinsic signal reflects the subcellular structure in an accurate way, SHG can be used for noninvasive high resolution structural imaging without exogenous labels in living muscle cells. This may be very important for detecting changes in myofibrillar organization occurring under pathophysiological conditions, e.g., in cardiac and skeletal myopathies. Due to the strong dependency of SHG on orientation and symmetries of the tissue, it may allow the study of dynamic interactions between the contractile proteins actin and myosin during force production and muscle shortening. Furthermore, SHG imaging can be combined with other nonlinear microscopical techniques, such as laser scanning multiphoton fluorescence microscopy, to simultaneously measure other dynamic cellular processes, representing a complementary method and extending the range of nonlinear microscopical methods.
We have used second harmonic generation (SHG) imaging to quantify
a strong intrinsic SHG-signal from cellular and subcellular muscle
fibre preparations. In isolated single muscle cells, the intrinsic
SHG-signal periodically follows the striation pattern and strongly
depends on the sarcomere length and the polarization of the
illuminating laser beam. At the subcellular level, the SHG signal
seems to be located mainly at the overlapping region of the (thin)
actin and (thick) myosin filaments. Thus, SHG imaging resolves the
arrangement of the contractile structures with high resolution
non-invasively and without chromophores. It may also allow to
study dynamic molecular interactions of the motor protein myosin
with actin filaments during force production and muscle
shortening.
We have developed an integrated microscopy system combining fast dual-excitation fluorescence photometry and digital image analysis with high spatial resolution, based mainly on standard components. With the combination of these well-established techniques in one setup it is possible to monitor intracellular calcium with both sufficiently high temporal and high spatial resolution on the same preparation for many biological applications. Our system consists of a commercially available dual-excitation photometric system, an attached intensified charge coupled device (ICCD) camera, and a frame grabber board. With this integrated setup one can easily switch between the fast photometric mode (vratio = 100 Hz) and the imaging mode (vratio = 4.l7 up to 17 Hz). We used the system to record Fura-2 calcium images (340/380 nm ratios), which were correlated with the faster spot measurements and were analyzed by means of image processing. As an example for its application we reconstructed caffeine-induced calcium transients released from the sarcoplasmic reticulum of isolated and permeabilized skeletal muscle fiber preparations. Such a combined technique will also be important for cellular studies using other fluorescence indicators. Additionally, the described system has an external trigger facility that enables combination with other cell physiological methods, e.g., electrophysiological techniques.
Fast photometric measurements and video-imaging of fluorescent indicators both are powerful tools in measuring the intracellular free calcium concentration of muscle and many other cells. as photometric systems yield a high temporal resolution, calcium imaging systems have high spatial but significantly reduced temporal resolution. Therefore we have developed an integrated system combining both methods and based mostly on standard components. As a common, sensitive Ca2+- indicator we used the fluorescent probe Fura-2, which is alternatingly excited for ratio measurements at 340/380 nm. We used a commercially available dual excitation photometric system (OSP-3; Olympus) for attaching a CCD-camera and a frame grabber board. To achieve the synchronization we had to design circuitries for external triggering, synchronization and accurate control of the filter changer, which we added to the system. Additionally, the software for a triggered image acquisition was developed. With this integrated setup one can easily switch between the fast photometric mode (ratio frequency 100 Hz) and the imaging mode (ratio frequency 4.17 Hz). The calcium images are correlated with the 25 times faster spot measurements and are analyzed by means of image processing. With this combined system we study release and uptake of calcium ions of normal and diseased skeletal muscle from mdx mice. Such a system will also be important for other cellular studies in which fluorescence indicators are used to monitor similar time dependent alterations as well as changes in cellular distributions of calcium.
Applying ultraviolet-laser microdissection enabled us to obtain very small myofibrillar preparations (e.g. 3 micrometers diameter) of normal and diseased muscle fibers which develop forces in the range of nano to micronewton too small to be measured with conventional force transducers. For the present study we built a very sensitive force transducer based on determining the force induced light beam deflection of a 50 micrometers optical fiber, connected to a 635 nm diode laser and detected by two photodiodes at two oppositely positioned 100 micrometers optical fibers to measure forces of smallest myofibrillar preparations. Also, for electrophysiological and stability studies selected vesicles of normal and myopathic fibers were trapped by the infrared-microbeam, brought into close membrane contact with other vesicles and fused by single pulses of the ultraviolet-microbeam. This approach allows for the first time to study membrane properties of normal and myopathic tissue in one preparation under the same intra and extravesicular medium conditions. These 'hybrid' vesicles should be of particular importance for studies of structural stability and electrophysiological properties or lateral mobility of ion channels, e.g. in presence or absence of the membrane-bound cytoskeleton dystrophin-glycoprotein complex which is less or not at all expressed in mdx-mice and Duchenne/Becker patients.
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