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Confocal microscopy studies of morphology and apoptosis: ovaries, limbs, embryos and insects
Citation:
ZUCKER, R. M. Confocal microscopy studies of morphology and apoptosis: ovaries, limbs, embryos and insects . Presented at Microscopy & Microanalysis, Ft. Lauderdale, FL, August 05 - 09, 2007.
Impact/Purpose:
presentation
Description:
Confocal laser scanning microscopy (CLSM) is a technique that is capable of generating serial sections of whole-mount tissue and then reassembling the computer-stored 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 problems of its own. One of the major problems using CLSM to image whole organs and embryos is tissue penetration of laser light. High quality morphological images begin by optimizing the sample preparation technique (1-4). 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. (5, 6) Confocal microscopy has been used by our laboratory to study cell death and morphology in embryos, ovaries, eyes, ears and limbs (1-4). The technique has revealed structural morphology and the initiation of cell death by the uptake LysoTracker dye into acidic cells (3). LysoTracker Red (LT) is a paraformaldehyde, fixable probe that 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 (1-4). 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 (RI) of the tissue within 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 confocal laser scanning microscopy instrument factors, allowed for the detection and visualization of apoptosis in fetal limbs and embryos which were approximately 500 microns thick. To show the versatility of this technique it has been applied to visualization of mosquito larvae. Mosquitoes were fixed with MEOH and stained with propidium iodide dehydrated in MeOH and cleared in BABB, similar to the mammalian tissues. Recently, spectroscopic imaging capacity has been incorporated into confocal microscopes from most confocal manufactures. Whereas classical confocal systems acquire a single wavelength data point determined by the characteristics of a band pass filter, these spectrophotometer systems use a wavelength dispersive spectrometer to acquire a series of wavelength data points that cover the entire spectral range. A dispersive prism (Leica SP) confocal microscope and a multichannel array (Zeiss 510 Meta) confocal microscope and one non confocal system (PARISS) were used to study the spectra derived from the apoptotic regions of the tissues. The LT spectra had a maximum peak around 610nm. The fixative, glutaraldehyde, had a maximum peak around 450nm and was added primarily to preserve the tissue morphology but provided fluorescence molecules in the green range that was useful to visualize morphology. Glutaraldehyde did not appear to interfere with the spectra of the LT. The understanding of the tissue spectra was extremely helpful in optimizing the staining protocol and helped clearly visualize the red LT apoptotic regions against a green morphological tissue background. The PARISS system was used to confirm the spectra derived from the two confocal systems. It had 1 nm resolution and was more accurate that the confocal spectral systems that resolution between 5 (Leica SP) and 10.7( Zeiss 510 Meta) nm resolution.