Whole insect and mammalian embryo imaging with confocal microscopy: Morphology and apoptosis

Abstract

Background:After fluorochromes are incorporated into cells, tissues, and organisms, confocal microscopy can be used to observe three‐dimensional structures. LysoTracker Red (LT) is a paraformaldehyde fixable probe that concentrates into acidic compartments of cells and indicates regions of high lysosomal activity and phagocytosis, which both correlate to apoptosis activity. LT has been shown to be an indicator of apoptotic cell death which is correlated to other standard apoptotic assays.Methods:The mammalian samples were stained with LT, fixed with paraformaldehyde/glutaraldehyde, dehydrated with methanol (MEOH), and cleared with benzyl alcohol/benzyl benzoate (BABB). Following this treatment, the tissues were nearly transparent. Mosquitoes were fixed with MEOH and stained with propidium iodide. Next the tissues were dehydrated with MEOH and cleared with BABB.Results:Tissues as thick as 500 μm can be visualized after clearing with BABB. LT staining revealed apoptotic regions in mammalian limbs, fetuses, and embryos. Morphological observation of insect tissue consisted of combining autofluorescence with either nucleic acid staining (either propidium iodide or ethidium bromide).Conclusions:The use of BABB matches the RI of the tissue within the suspending medium. It helps in increasing the penetration of laser light in a confocal microscope by reducing the amount of light scattering artifacts and allows for the visualization of morphology in thick tissues. LT is a probe that stains the acid regions of tissues and cells and has been correlated to apoptosis. Morphological features of a tissue or organism (embryo, mosquito larvae) can be elucidated by fixation aldehydes, autofluorescence, and red‐emitting probes. This sample preparation procedure with optimization of confocal laser scanning microscopy allowed for the detection and visualization of apoptosis in fetal limbs and embryos which were ∼500‐μm thick. © 2006 International Society for Analytical Cytology

An automated approach for three-dimensional quantification of fibrillar structures in optically cleared soft biological tissues

“…Specifically, we combine tissue clearing using a solution of benzyl alcohol to benzyl benzoate (BABB) [26,27] with SHG imaging of fibrillar collagen [28,29], Fourier-based image analysis for characterizing collagen organization [30 -32] and maximum-likelihood estimation (MLE) for distribution fitting of three-dimensional-orientational datasets [33]. First, our method yields the three-dimensional distribution of amplitudes (18 resolution), representing the orientations of fibrillar collagen extracted from a z-stack; second, it identifies isotropic regions in the tissue where no preferred fibre orientations are observed; third, it identifies regions of anisotropy characterized by two structural parameters: one describing the preferred (or principal) orientation of the fibres (denoted m), and the other describing the degree of fibre alignment (or concentration) about that preferred orientation (denoted b).…”

Altered health outcomes in adult offspring of Sprague Dawley and Wistar rats undernourished during early or late pregnancy

“…Following decapsulation, kidneys were sliced sagittally and fixed overnight at 371C in freshly prepared 4% paraformaldehyde. Fixed kidneys were dehydrated to 70% methanol, stained with 10 mM Yo-Pro-1 (Molecular Probes, Eugene, OR), further dehydrated to 100% methanol, and cleared using a solution of benzyl alcohol and benzyl benzoate as previously described (Zucker, 2006). Cleared kidneys in benzyl benzoate were mounted between glass cover slips sealed to aluminum microscope slides with a circular hole in the center.…”

Reliability of confocal microscopy spectral imaging systems: Use of multispectral beads

“…Decreased fluorescence due to slider problems resulted in higher PMT settings and more averaging (33). Although it is conceivable that these data could have been derived from many types of samples having fluorescence emissions between 500 and 700 nm, we feel that having defined particles with known spectral patterns has made the task easier and more reproducible to achieve.…”

“…It is suggested that investigators use only PMT 2 with sequential scanning of the fluorescence emission instead of simultaneous scanning of the multiple excitation probes on a SP1 system until the problem is resolved. This was done to acquire two fluorescent signals sequentially into one PMT from a sample (33).…”

“…That will create noisy images which can only be improved by excessive averaging or better sample preparation. In fact due to a perceived lack of fluorescence in PMT 1, we changed our sample preparation protocol of ovaries, and embryos to actually introduce more fluorescence into the tissue by fixing with glutaraldehyde molecules (17,33). The sensitivity of a system will be decreased, as fewer photons enter the PMT.…”