The plasma membrane of various mammalian cell types is heterogeneous in structure and may contain microdomains, which can impose constraints on the lateral diffusion of its constituents. Fluorescence correlation spectroscopy (FCS) can be used to investigate the dynamic properties of the plasma membrane of living cells. Very recently, Wawrezinieck et al. (Wawrezinieck, L., H. Rigneault, D. Marguet, and P. F. Lenne. 2005. Biophys. J. 89:4029-4042) described a method to probe the nature of the lateral microheterogeneities of the membrane by varying the beam size in the FCS instrument. The dependence of the width of the autocorrelation function at half-maximum, i.e., the diffusion time, on the transverse area of the confocal volume gives information on the nature of the imposed confinement. We describe an alternative approach that yields essentially the same information, and can readily be applied on commercial FCS instruments by measuring the diffusion time and the particle number at various relative positions of the cell membrane with respect to the waist of the laser beam, i.e., by performing a Z-scan.

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To assess the risk of femoral tunnel convergence in combined anterior cruciate ligament (ACL) and anterolateral ligament (ALL) reconstructions. The hypothesis was that a more proximal and anterior orientation of the ALL femoral tunnel should reduce the risk of convergence with the ACL femoral tunnel. MethodsFifteen fresh-frozen cadaver knees were examined. An anatomic ACL femoral tunnel was drilled arthroscopically in each specimen and ALL tunnels were made in two directions: 1) 0° coronal angulation and 20° axial angulation , 2) 30° coronal angulation and 30° axial angulation. Computed tomography scans were performed to investigate tunnel convergence and to measure the minimal distance between tunnels, tunnel length and the LFC width. ResultsTunnel convergence occurred in 20 of 30 cases (67%). Convergence was significantly reduced when tunnels were drilled at 30° coronal and 30° axial angulation (P<0.05). The mean length of the ALL tunnel was 15.9mm (95% CI [13.6; 18.1]) and was independent of ALL tunnel angulation. The mean minimal distance between the ALL and ACL tunnel was 3.1 mm (95% CI [2.1; 4.1]). The odds ratio for tunnel convergence was 3.5 for small LFC, relative to large LFC (n.s.) ConclusionA high risk of tunnel convergence was observed when performing combined ACL and ALL reconstructions. The clinical relevance of this work is that the occurrence of tunnel conflicts can be reduced by aiming the ALL tunnel in a more proximal and anterior direction. Surgeons should be aware of this, since tunnel convergence could jeopardize the ACL reconstruction and fixation.

The heterogeneity in composition and interaction within the cellular membrane translates into a wide range of diffusion coefficients of its constituents. Therefore, several complementary microfluorimetric techniques such as fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP) and singleparticle tracking (SPT) have to be applied to explore the dynamics of membrane components. The recently introduced raster image correlation spectroscopy (RICS) offers a much wider dynamic range than each of these methods separately and allows for spatial mapping of the dynamic properties. RICS is implemented on a confocal laser-scanning microscope (CLSM), and the wide dynamic range is achieved by exploiting the inherent time information carried by the scanning laser beam in the generation of the confocal images. The original introduction of RICS used two-photon excitation and photon counting detection. However, most CLSM systems are based on one-photon excitation with analog detection. Here we report on the performance of such a commercial CLSM (Zeiss LSM 510 META) in the study of the diffusion of the fluorescent lipid analog 1,1 0 -dioctadecyl-3,3,3 0 ,3 0 -tetramethyl-indodicarbocyanine perchlorate (DiI-C 18 (5)) both in giant unilamellar vesicles and in the plasma membrane of living oligodendrocytes, i.e., the myelin-producing cells of the central nervous system. It is shown that RICS on a commercial CLSM with analog detection allows for reliable results in the study of membrane diffusion by removal of unwanted correlations introduced by the analog detection system. The results obtained compare well with those collected by FRAP and FCS.