Fluorescent stains for quantification of DNA by confocal laser scanning microscopy in 3-D.

Confocal microscopy requires the use of fluorophores to visualize structures of interest within a specimen. To perform reliable measurements of the intensity of fluorescence, the stain should be specific, penetrate well into tissue sections, and bind stoichiometrically. Furthermore, emission must be linear with respect to DNA content and brightness, and fluorescence should be stable. Confocal microscopy is used to determine DNA ploidy and to analyze texture of nuclei, which is accomplished in three dimensions, because nuclei can be measured within the original tissue context. For this purpose the sample must be stained with a DNA binding fluorophore with the properties described above. Stains with different properties have been developed for different applications. We review here the advantages and disadvantages of these different stains for analyzing DNA ploidy and nuclear texture using three-dimensional microscopy. We conclude that SYBR green I and TO-PRO-3 are the most suitable stains for this purpose at present.

Accurate measurement of the dynamic response of a scanning electronic portal imaging device.

An important condition for the safe introduction of dynamic intensity modulated radiotherapy (IMRT) using a multileaf collimator (MLC) is the ability to verify the leaf trajectories. In order to verify IMRT using an electronic portal imaging device (EPID), the EPID response should be accurate and fast. Noninstantaneous dynamic response causes motion blurring. The aim of this study is to develop a measurement method to determine the magnitude of the geometrical error as a result of motion blurring for imagers with scanning readout. The response of a liquid-filled ionization chamber EPID, as an example of a scanning imager, on a moving beam is compared with the response of a diode placed at the surface of the EPID. The signals are compared under the assumption that all EPID rows measure the same dose rate when a straight moving field edge is imaged. The measurements are performed at several levels of attenuation to investigate the influence of dose rate on the response of the detector. The accuracy of the measurement method is better than 0.25 mm. We found that the liquid-filled ionization chamber EPID does not suffer from significant motion blurring under clinical circumstances. Using a maximum gradient edge detector to determine the field edge in an image obtained by a liquid-filled ionization chamber EPID, errors smaller than 1 mm are found at a dose rate of 105 MU/min and a field edge speed of 1.1 cm/s. The errors reduce at higher dose rates. The presented method is capable of quantifying the geometrical errors in determining the position of the edge of a moving field with subpixel accuracy. The errors in field edge position determined by a liquid-filled ionization chamber EPID are negligible in clinical practice. Consequently, these EPIDs are suitable for geometric IMRT verification, as far as dynamic response is concerned.

Leaf position verification during dynamic beam delivery: a comparison of three applications using electronic portal imaging.

The use of a dynamic multileaf collimator (MLC) to deliver intensity-modulated beams presents a problem for conventional verification techniques. The use of electronic portal imaging to track MLC leaves during beam delivery has been shown to provide a solution to this problem. An experimental comparison of three different verification systems, each using a different electronic portal imaging technology, is presented. Two of the systems presented are commercially available imagers with in-house modifications, with the third system being an in-house built experimental system. The random and systematic errors present in each of the verifications systems are measured and presented, together with the study of the effects of varying dose rate and leaf speed on verification system performance. The performance of the three systems is demonstrated to be very similar, with an overall accuracy in comparing measured and prescribed collimator trajectories of approximately +/-1.0 mm. Systematic errors in the percentage delivered dose signal provided by the accelerator are significant and must be corrected for good performance of the current systems. It is demonstrated that, with suitable modifications, commercially available portal imaging systems can be used to verify dynamic MLC beam delivery.