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.
We report an application of the combined third order microscopy techniques to reveal structure and morphology of the
peripheral nerve in mice. The resonant Coherent Anti-Stokes Raman Scattering (CARS) and third harmonic generation
(THG) techniques have been applied to visualize structure of the myelinated peripheral axon. While CARS was quite
efficient in selective imaging of the cladding layer via characteristic Raman active vibrations of dense lipid structures
constituting the layers, the THG microscopy helped to clearly reveal the degree of optical and nonlinear optical
inhomogeneity of the axon core (that may have further important implications).
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 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