[Show abstract][Hide abstract]ABSTRACT: Recent advances in high-resolution imaging have provided valuable novel insights into structural relationships within cells and tissues both in vitro and in vivo. An analy-sis of this kind is regularly done by optical sectioning using either confocal or deconvolution mi-croscopy. However, the reconstruction of 3D images suffers from light scattering and absorption with increasing depth by finite transparency of the used media. Photobleaching of fluorochromes has been especially troublesome and often the only remedy for loss of signal during optical sec-tioning is to reduce the number of sections. This causes disparities in the x–y and z dimensions of voxels, which lead to vertical distortion of the original stack of images and necessitates interpola-tion. Interpolation is necessary to fill up the gaps between consecutive sections in the original image stack to obtain cubic voxels. The present manuscript describes a novel method for adaptive compensation of attenuation of light intensity in stacks of fluorescence microscopy images that is based on a physical model of light attenuation. First, we use a fast interpolation technique to generate a cubic voxel-based volume stack with the aid of a contribution look up table. With the contribution look up table, multiple calculations are avoided, which substantially reduces the computational time without compromising the accuracy of the restoration procedure. Second, each section within the resulting volume is processed to rectify its intensity values that have been altered due to photobleaching and scattering and absorption. The method allows to define the last good section in the stack and the correction is then done automatically. Microsc. Res. Tech. 71:146–157, 2008. V V C 2007 Wiley-Liss, Inc. INTRODUCTION Developments in fluorescence microscopy and the availability of fluorescently labelled antibodies and probes for localization of molecules and organelles have made the microscope an indispensable tool with which one can map specific molecules to subcellular loci. Furthermore, temporal kinetics of the distribution of a molecule of interest can be followed in time with variable sampling frequency (Farkas et al., 1993; Zhang et al., 2002). Collectively, fluorescence micros-copy has evolved into a versatile and quantitative tool allowing deeper insight into cell and organelle biology (De Giorgi et al., 1996; Taylor and Wang, 1980). As living cells and tissues are three-dimensional (3D) structures, the spatial relationship among their components is often at issue. However, an analysis of this kind is hampered by an image of a specimen being degraded by out of focus information from planes below and above the current focal plane. This problem has been partly alleviated (White et al., 1987) by confocal microscopy. In a confocal microscope, the specimen is imaged by a combination of point illumination and point detection, which eliminates the bulk of out-of-focus information, thus allowing effective serial optical sectioning of a specimen (Laurent et al., 1992). The coupling of the confocal microscope with computer technology has greatly enhanced the capabilities of the microscope, allowing further video-enhancement and analysis of the image obtained (Eils and Athale, 2003). As a result, analysis of the three dimensional architec-ture of cells, which cannot be accomplished by conven-tional light microscopy, is now possible (Gerlich et al., 2001; Shotton and White, 1989). Three dimensional confocal images may have differ-ent resolutions in the x, y, and z directions and hence the dimensions of voxels may vary in each direction. Generally, the scan interval in the z direction is larger than that in the other two directions. The intensity values of voxels are likely to vary discontinuously to a large degree along the z direction, which will affect the post-processing and analysis. Hence, to generate cubic voxels, it is necessary to fill the gaps between consecutive optical sections by first using an
[Show abstract][Hide abstract]ABSTRACT: Recent development in high-resolution confocal imaging has provided valuable novel insights into struc- tural relationships within cells and tissues in vitro and in vivo. Advancement in volume visualization tech- nique enables real-time visualization of 3D dataset collected with confocal microscope. The stereo display of the reconstructed D model provides an intuitive way for users to view and manipulate the 3D cellular environment, thus the information extraction and quantitative analysis on the 3D cellular volume are sim- plified.
[Show abstract][Hide abstract]ABSTRACT: Recent advancement in high-resolution confocal imaging has provided valuable novel insights into structural relationships within cells and tissues in vitro and in vivo. Development in volume rendering technique enables visualization of 3D dataset in real-time. Here, we present a system in which 3D models are reconstructed from the stacked 2D images obtained with a confocal microscope, and subsequently visualized using volume rendering technique. Using the reconstructed 3D model, we apply virtual reality method to provide an intuitive way to view and manipulate the 3D cellular environment.