Confocal Microscopy of Thick Tissue Sections: 3D Visualization of Rat Kidney Glomeruli.

Abstract

Confocal laser scanning microscopy (CLSM) is a technique that is capable of generating serial sections of whole-mount tissue and then reassembling the computer acquired images as a virtual 3-dimensional structure. In many ways CLSM offers an alternative to traditional sectioning approaches. However, the imaging of whole-mount tissues presents technical limitations of its own. One of the major problems using CLSM to image whole organs and embryos is the penetration of laser light into the tissue.. High quality morphological images begin by optimizing the sample preparation technique (1-2). Additional factors include evaluating CLSM performance by optimizing the acquisition variables (i.e. objective lens, averaging, pinhole size, bleaching, PMT voltage, laser excitation source, and spectral registration) of the confocal microscope. (3-4) Confocal microscopy has been used by our laboratory to study cell death and morphology in embryos, ovaries, eyes, ears, and limbs (1-2). The technique has revealed structural morphology and visualized areas of cell death by the uptake of the LysoTracker dye into phagolysomes. LysoTracker Red (LT) is fixable by paraformaldehyde and concentrates in acidic compartments of cells. In whole tissues, this accumulation indicates regions of high lysosomal activity and phagocytosis. LT staining is an indicator of apoptotic cell death and correlates with other standard apoptotic assays (1-2). LT staining revealed cell death regions in mammalian limbs, neonatal ovaries, fetuses and embryos. The mammalian samples were stained with LT, fixed with paraformaldehyde/glutaraldehyde, dehydrated with methanol (MeOH), and cleared with benzyl alcohol/benzyl benzoate (BABB). The use of BABB matches the refractive index of the tissue to that of the suspending medium. BABB helps increase the penetration of laser light during CLSM by reducing the amount of light scattering artifacts and allows for the visualization of morphology in thick tissue. Following this treatment, the tissues were nearly transparent. This sample preparation procedure, combined with the optimization of CLSM instrument factors, allowed for the detection and visualization of apoptosis in fetal limbs and embryos which were approximately 500 microns thick.Spectroscopic imaging capacity has been incorporated into all manufacturers' confocal microscopes. The LT spectra had a maximum peak around 610nm while the fixative, glutaraldehyde (Glut), had a maximum peak around 450nm. Glut was added primarily to preserve the tissue morphology, but also provided molecules emitting in the green fluorescence range that helped to visualize the morphology of the tissue. The understanding of the spectra derived from the tissue was extremely useful in optimizing the staining protocol. We have continued to incrementally improve the tissue staining and sample preparation techniques to achieve better quality 3D images.