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Confocal microscopy of thick tissue sections: 3D visualizaiton of rat kidney glomeruli
ZUCKER, R. M., J. M. ROGERS, AND R. G. ELLIS-HUTCHINGS. Confocal microscopy of thick tissue sections: 3D visualizaiton of rat kidney glomeruli. Presented at Pre M*M Meeting: Cellular Analysis - Linking Quantitation to Structure and Function, Albuquerque, NM, August 02 - 03, 2008.
Presentation @ Pre - Microscopy & Microanalysis 2008 Meeting
Confocal laser scanning microscopy (CLSM) as a technique capable of generating serial sections of whole-mount tissue and then reassembling the computer-acquired images as a virtual 3-dimentional structure. In many ways CLSM offers an alternative to traditional sectioning approaches. However, the imaging of such whole-mounts presents technical problems of its own. One of the major problems with using CLSM to image whole organs and embryos is the penetration of laser light into tissue. High quality morphological images begin 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. Confocal microscopy has been used by our laboratory to study cell death and morphology in embryos, ovaries, eyes, ears, and limbs. The techniques 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. 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 610 nm while the fixative, glutaraldehyde (Glut), had a maximum peak around 450 nm. 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 preparations techniques to achieve better quality 3D images. Recently we have applied this technique to observe glomeruli in kidneys. By staining with YoPro-1, we have been able to visualize and count glomeruli using Imaris software in thick kidney tissue (600 microns) derived from a postnatal day 22 rat. Currently we are evaluating the data from rats treated with chemicals or undernourished during pregnancy to establish what advantages this technique offers over the exhaustive stereology method, the current standard for glomeruli quantification Spectroscopic imaging of kidney features was conducted and comparisons made using a PARISS spectrograph, Leica SP1, Nikon C1Si and a Zeiss Meta 510. From this data we gained insight regarding the selective staining of glomeruli by YoPro-1. The spectra derived form these different confocal spectral machines all showed that YoPor-1 selectively stained glomeruli in the 500-530 range in a positive way and in a negative way between 550-650 nm. The YoPro-1 spectra had a maximum peak around 525 nm while the kidney tubule background had increased fluorescence intensity at longer wavelengths. Achieving optimal contrast between the glomeruli and surrounding tubules has allowed us to visualize the glomeruli as 3D spots in a solid tissue up to 1 mm thick. The Imaris “spots” procedure utilizes contrast between the background and the round structure of interest for correct identification. The understanding of the spectra derived from the tissue has been extremely useful in setting up the image acquisition for optimized glomeruli quantification using Imaris. The image acquisition procedure has been optimized by measuring only the spectra between 500-530 using a bandpass filter, having near-saturation intensities and eliminating the fluorescence spectra above the 530 nm wavelength range. If this fluorescence spectrum from the longer wavelengths is included in the 3D tissue stack, then artifacts in glomeruli counting spots procedure increases due to a loss in contrast associated with the increase in tubule background fluorescence. Not using a green long pass filter to acquire this glomeruli data seems contradictory to normal confocal logic, which tends to acquire all the photons possible. We have also evaluated various hardware parameters which affect our image acquisition. These include the type of objectives, numerical aperture of objectives, confocal microscope vendors, zoom factor, pinhole size, averaging, intensity compensation, z step size, PMT voltage and laser power. Quality assurance of the confocal microscope is necessary for this quantification to be successful and these factors will be discussed in the presentation. We have continued to incrementally improve the YoPro staining of glomeruli tissue and sample preparations techniques to achieve better quality 3D images.