A large field CCD system for quantitative imaging of microarrays.

We describe a charge-coupled device (CCD) imaging system for microarrays capable of acquiring quantitative, high dynamic range images of very large fields. Illumination is supplied by an arc lamp, and filters are used to define excitation and emission bands. The system is linear down to fluorochrome densities <<1 molecule/microm2. The ratios of the illumination intensity distributions for all excitation wavelengths have a maximum deviation approximately +/-4% over the object field, so that images can be analyzed without computational corrections for the illumination pattern unless higher accuracy is desired. Custom designed detection optics produce achromatic images of the spectral region from approximately 450 to approximately 750 nm. Acquisition of a series of images of multiple fluorochromes from multiple arrays occurs under computer control. The version of the system described in detail provides images of 20 mm square areas using a 27 mm square, 2K x 2K pixel, cooled CCD chip with a well depth of approximately 10(5) electrons, and provides ratio measurements accurate to a few percent over a dynamic range in intensity >1000. Resolution referred to the sample is 10 microm, sufficient for obtaining quantitative multicolor images from >30,000 array elements in an 18 mm x 18 mm square.

Energy expenditure in lean and obese prepubertal children.

The relationship between energy expenditure and obesity was examined in prepubertal children. Consenting fifth graders underwent Tanner Staging, weight, height and skinfold measurements. Subjects were selected for further study to obtain equal numbers of girls and boys with a wide range of body composition. Weight, total daily energy expenditure (TDEE) by doubly labeled water (DLW), resting metabolic rate (RMR), and body composition were measured. Children were grouped into level of obesity based on tertiles of subscapular plus triceps skinfolds. The skinfold tertiles did quite well in grouping subjects by degree of obesity, as differences in percent fat in each tertile were significantly different. There were no differences in fat-free mass between the groups, while the highest tertile group weighed 14 kg more than the lowest. For DLW, energy expenditure was calculated using day 8 and day 9 urine samples as the final time point to examine precision. Mean energy expenditure using either day was nearly identical (2220 +/- 400 vs 2300 +/- 370 kcal/d), with a CV of the difference of 5.5%. No differences in RMR, energy expended in activity, or TDEE between the three groups were observed. A reduction in RMR or TDEE could not explain differences in obesity in these prepubertal children. However, the fact that the heaviest children expended the same amount of energy in activity and had the same TDEE as the leanest, while weighing 14 kg more, indicates that the obese children had a reduced activity level.