No abstract

The days of being able to ascertain instrument performance by simply peering through the eye pieces at a specimen are gone. However, users and granting agencies need to be confident that data collected on these instruments is uniform and quantifiable both over time and between instruments. Ideally, a LASER should not fluctuate, illumination should be completely uniform, and colors should be perfectly aligned. To check the current performance of imaging equipment, we conducted a worldwide research study utilizing three image-based tests: long-/short-term illumination stability, co-registration of signals across various wavelengths, and field illumination uniformity. To differentiate between "acceptable" and "unacceptable" performance, the deviation in illumination power could not exceed 10% (long term) or 3% (short term), the difference in the center-of-mass of imaged multicolored beads could not exceed >1 pixel between different wavelengths, and field illumination values could not exceed 10% (horizontal) or 20% (diagonal) deviation. This study established the current state of microscope performance through simple, efficient, and robust tests, while defining relative standards to assist cores in maintaining their instruments in optimal operating conditions. We developed cross-platform performance standards that will improve the validity of quantitative measurements made using various light microscopes.

As part of an ongoing effort to increase image reproducibility and fidelity in addition to improving cross-instrument consistency, we have proposed using four separate instrument quality tests to augment the ones we have previously reported. These four tests assessed the following areas: (1) objective lens quality, (2) resolution, (3) accuracy of the wavelength information from spectral detectors, and (4) the accuracy and quality of spectral separation algorithms. Data were received from 55 laboratories located in 18 countries. The largest source of errors across all tests was user error which could be subdivided between failure to follow provided protocols and improper use of the microscope. This truly emphasizes the importance of proper rigorous training and diligence in performing confocal microscopy experiments and equipment evaluations. It should be noted that there was no discernible difference in quality between confocal microscope manufactures. These tests, as well as others previously reported, will help assess the quality of confocal microscopy equipment and will provide a means to track equipment performance over time. From 62 to 97% of the data sets sent in passed the various tests demonstrating the usefulness and appropriateness of these tests as part of a larger performance testing regiment.

No abstract

Forces that elongate the spindle during anaphase B of mitosis might be generated in the asters, in the spindle, or in both. In the fungus Nectria haematococca, it has already been shown that the asters pull on the spindle pole bodies (SPBs) throughout anaphase B. In this study, we used computerized video motion analysis to characterize brief episodes of spindle bending and straightening to find out if such bending is caused by spindle pushing forces. In three episodes there were two distinct components of spindle bending and straightening: one spanning the entire episode and comprising spindle elongation and another, superimposed on the first, involving a shortening of the distance between the SPBs. In a fourth episode, only spindle elongation was involved. All four spindles elongated rapidly while bending and underwent net growth during the overall bending-straightening episode at an average rate of 4.2 p d m i n . The path of one aster of a fifth mitotic apparatus was blocked by a large, occluding vacuole. This obstacle caused the migration of the mitotic apparatus to stop, resulting in a long (25 sec) episode of spindle curving and bending, usually without any substantial reduction in the distance between the SPBs as well as a marked reduction (from 4.7 to 0.65 p d m i n ) in the rate of spindle elongation. The results provide evidence that spindle pushing forces are active in vivo during anaphase B in N . haematococca and that they, along with astral pulling forces, help to elongate the spindle at a mostly constant rate. This is the first demonstration of both kinds of spindle elongation forces in the same organism.