Automatic section thickness determination using an absolute gradient focus function.

Quantitative analysis of microstructures using computerized stereology systems is an essential tool in many disciplines of bioscience research. Section thickness determination in current nonautomated approaches requires manual location of upper and lower surfaces of tissue sections. In contrast to conventional autofocus functions that locate the optimally focused optical plane using the global maximum on a focus curve, this study identified by two sharp 'knees' on the focus curve as the transition from unfocused to focused optical planes. Analysis of 14 grey-scale focus functions showed, the thresholded absolute gradient function, was best for finding detectable bends that closely correspond to the bounding optical planes at the upper and lower tissue surfaces. Modifications to this function generated four novel functions that outperformed the original. The 'modified absolute gradient count' function outperformed all others with an average error of 0.56 µm on a test set of images similar to the training set; and, an average error of 0.39 µm on a test set comprised of images captured from a different case, that is, different staining methods on a different brain region from a different subject rat. We describe a novel algorithm that allows for automatic section thickness determination based on just out-of-focus planes, a prerequisite for fully automatic computerized stereology.

UVB induced photooxidation of vitamin E.

The photochemistry of alpha-tocopherol (alpha-TH, vitamin E) may contribute to its inhibition of UVB (290-320 nm) photocarcinogenesis. Photochemical reactions of alpha-TH were studied by monitoring the fate of alpha-TH in UVB irradiated liposomes and solution. Soy phosphatidylcholine (SPC) and dioleoylphosphatidylcholine (DOPC) liposomes were supplemented with alpha-TH (1.0 mol % alpha-TH/phospholipid) and irradiated with UVB at a dose rate of 6.0 J m-2s-1 for up to 90 min. alpha-TH was rapidly depleted in UVB irradiated liposomes. Oxidative damage, assessed by monitoring lipid peroxidation, was suppressed in SPC liposomes until alpha-TH was depleted to 20% of initial levels. alpha-TH also was rapidly depleted by UVB irradiation in acetonitrile/H2O (4:1 v/v) solution. In SPC liposomes, products previously identified as marker products for peroxyl radical scavenging by alpha-TH were observed, including alpha-tocopherol quinone, 5,6-epoxy-alpha-tocopherol quinone, and 2,3-epoxy-alpha-tocopherol quinone. These products also were formed in DOPC liposomes, which are resistant to lipid peroxyl radical formation. In addition, an alpha-tocopherol dihydroxy dimer and several 8a-(hydroperoxy)epoxytocopherones were identified by HPLC and HPLC-MS. The dimer appears to result from recombination of photoinduced tocopheroxyl radicals. Products associated with peroxyl radical scavenging (quinones, epoxyquinones, 8a-(hydroperoxy)epoxytocopherones) and with UVB dependent production of tocopheroxyl radicals (dihydroxy dimer) also were found when alpha-TH was oxidized by UVB in acetonitrile. Because the acetonitrile contained no autoxidizable substrate, formation of peroxyl radical derived products may occur via intermediate tocopherone peroxyl radicals. These results indicate that alpha-TH photooxidation proceeds via competing reactions of UVB induced tocopheroxyl radicals.