Confocal microscopy three dimensional reconstruction

Both the light microscope (LM) and the confocal scanning laser microscopy (CLSM) operate in the micron range. The advantage of the CLSM is that it allows for measurements of dynamic events such as crack propagation and three-dimensional reconstructions. Moreover, the optical resolution in the CLSM is about 40% better than in the LM. The CLSM also makes it possible to study bulk samples with a minimum of preparation, which is quite important when handling multiphase colloidal structures such as emulsions. 

Fig. 4. Combined use of flow cytometry/cell sorting and confocal laser scanning microscopy. TNF-a/ActD treated Hepa-1 cells were stained with CMX Rosamine and then analyzed for mitochondrial membrane potential and NAD(P)H fluorescence (A). Cells in different regions of the cytogram were then sorted, and subsequently stained with the DNA fluorochrome Hoechst 33342. These cells were then examined by CLSM. (B), (C), and (D) show three-dimensional reconstructions of nuclei from cells sorted from healthy, early apoptotic, and late apoptotic populations. Whereas healthy cells show normal round nuclei, early and late apoptotic cells show progressive chromatin condensation/margination and nuclear fragmentation.

Many of the comments made regarding confocal microscopy can also be made regarding X-ray microtomography, in that a full three-dimensional reconstruction of the scanned sample results from the analysis. Thus, fractal analysis can be made by point-to-point correlations in the three-dimensional structure or within two-dimensional slices and then applying the codimension rule. 

In the last decade confocal laser scanning microscopy (CLSM) was shown to be a helpful tool for various further tasks of microparticle characterization (Lamprecht et al., 2000a, b, c). It minimizes the light scattered from out-of-focus structures, and permits the identification of several compounds through use of different fluorescence labels. Therefore, CLSM can be applied as a non-destructive visualization technique for microparticles. Moreover, CLSM allows visualization and characterization of structures not only on the surface, but also inside the particles, provided the carrier matrices are sufficiently transparent and can be fluorescently labeled by collecting several coplanar cross-sections, a three-dimensional reconstruction of the inspected objects is possible. Figure 6.13 shows the application of CLSM to investigatation of the cross-sectional structures of spray-dried powders of maltodextrin (MD) with a dextrose equivalent value of DE = 2 and 20. Florescein sodium salt was dissolved in the feed solution as a fluorescent probe of the carrier... 

Andjelkovic AV, Zochowski MR, Morgan F et al (2001) Qualitative and quantitative analysis of monocyte transendothelial migration by confocal microscopy and three-dimensional image reconstruction. In Vitro Cell Dev Biol Anim 37 111-120... 

The technique of confocal microscopy can be considered as optical sectioning. A three-dimensional (3D) image is obtained by reconstructing a deck of plane images. Figure 1.47... 

Confocal Microscopy A microscopic technique used to produce three-dimensional images of specimens that actually have considerable depth. A series of shallow depth-of-field image slices through a thick specimen are obtained. The three-dimensional image is then obtained by reconstruction so that no out-of-focus elements contribute to the final image. 

Scivetti M, PiloUi GP, Corsahni M, Lucchese A, Favia G. Confocal laser scanning microscopy of human cementocytes analysis of three-dimensional image reconstruction. Ann Anat 2007 189 169-74. 

Visualization of serial sections is the most frequently used computer technique in confocal microscopy, independent of specific applications [83], [108]. [109], Most commercial instruments have three-dimensional software implementations ready for the user. Three-dimensional objects are displayed on the computer screen by staking up the individual sections. Such techniques have been reviewed in several articles [110]-[114]. Besides surface rendering based on contours, volumetric representations that rely on the intensities of the voxels are the preferred techniques in confocal microscopy. A comparative assessment of both techniques is given in [115]. Reconstructions are presented three-dimensionally on a computer graphics screen by using either visual cues, animation, of stereoscopic displays. 

J. A. Conchello and E. W. Hansen, Enhanced 3-D reconstruction from confocal scarming microscope images. I. Deterministic and maximum likelihood reconstructions, Appl. Opt. v., 1990, 29, 3795-3804 see also J. Markham and J.-A. Conchello, Fast maximum- likelihood image-restoration algorithms for three dimensional fluorescence microscopy. J. Opt. Soc. Am. A, V. 18, (2001) p. 1062. 

---