When developing optical systems, every element within the light path is considered in technical optics. In ophthalmic optics, however, spectacle lenses are usually designed to provide a given optical power at a position called vertex sphere, ignoring the actual imaging processing inside the eye. We have developed a novel technology (trade name DNEye® PRO) overcoming this practice. The computation of the wavefronts does not stop at the back surface of the spectacle lens but is continued right into the eye through its refracting surfaces. The assessment no longer takes place at the vertex sphere, but at the retina. This calculation is based on individual measurements of biometrical parameters of the eye and comprises the complex shapes of the wavefronts and of the refracting surfaces including their higher-order components. As a result, effects which arise from the individual structure of the eye and its components are considered giving rise to sharper imaging and better design retention.
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.
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