Confocal microscopy, apoptosis

Zucker, R.M.Technical note Whole insects and Mammalian Embryo Imaging with Confocal Microscopy Morphology and Apoptosis. Cytometry 2006 69A 1143- 1152 (COVER). 

Lemasters JJ, QianT, Elmore SP,Trost LC, Nishimura Y, Herman B, Bradham CA, Brenner DA, and Nieminen AL. Confocal microscopy of the mitochondrial permeability transition in necrotic cell killing, apoptosis and autophagy. BioFactors (Oxford, England) 8 283-285,1998. 

Mitochondrial parameters Flow cytometry and confocal microscopy Fast large cell numbers Early stages of apoptosis Multiparameter option Indirect early stage changes may not be definitive proof... 

Many of these dyes and their applications have been reviewed in detail elsewhere (50). Of note, probes are available for both UV and 488 nm excitation and many of these calcium probes (Fura-l, Indo-1, Calcium Green-2) have the advantage of utilizing a ratiometric fluorescence read-out. These dyes have been successfully used in multiparametric analyses, using both flow cytometry and confocal microscopy, which has led to an enhanced understanding of the role of [Ca2+]i in apoptosis. Burchiel et al. (51) have recently published a review of multiparametric flow cytometric Ca2+ analysis. 

As with flow cytometry, multiparameter apoptosis assays may also be performed by confocal laser scanning microscopy (CLSM). Using the approach similar to that detailed above for flow cytometry, we have examined NADPH content, mitochondrial membrane potential (CMX Rosamine fluorescence), and mitochondrial mass (Mitotracker Green), by CLSM. Figure 3 shows an example of a typical multiparameter assay performed by confocal microscopy. 

AIF is an apoptotic effector protein that induces chromatin condensation and DNA fragmentation [51], Confocal microscopy (Fig. 9) reveals that AIF is strictly confined to mitochondria and thus colocalizes with mtHSP70 in control cells (Fig. 9a). In early apoptosis, AIF translocates to the nucleus, but in late apoptosis (with fragmented chromatin), immunolabeling moves back in the cytoplasm again in mitochondria [52-54], After treatment with cisPt (Fig. 9), AIF and mtHSP70 colocalize in clusters within the perinuclear cytoplasm. 

Zucker, R.M. and Jeffay SC. Confocal Laser Scanning Microscopy of Whole Mouse Ovaries Excellent Morphology with Apoptosis Detection and Spectroscopy. Cytometry 2006 69A August 2006 930-939 (COVER). 

Gatti, R. Belletti, S. Orlandini, G. Bussolati, O. Dair Asta, V Gazzola, G. C. Comparison of annexin V and calcein-AM as early vital markers of apoptosis in adherent cells by confocal laser microscopy. J. Histochem. Cytochem. 1998, 46, 895-900. 

Davis, W. P Janssen, I. M. W Mossman, B. T. Taatjes, D. J. Simultaneous triple fluorescence detection of mRNA localization, nuclear DNA, and apoptosis in cultured cells using confocal scanning laser microscopy. Histochem. Cell Biol. 1997, 108, 307-311. 

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