This protocol was developed to allow dosimetry measurements to be performed as close as possible to real cell irradiation conditions for radiobiological studies. With this protocol, we can determine the exact dose received by cells making it applicable to many x-ray facilities and can take into account all of the parameters and translate to dosimetry. All irradiation parameters must be set upstream to allow an optimal acquisition of the dosimetry measurement requiring a close collaboration between physicists and radiobiologist.
To perform an irradiation field evaluation, place a self-developing dosimetry film onto the support used for irradiation and irradiate the film with at least two grays to obtain a well-marked irradiation field. Scan the self-developing dosimetry film using a dedicated scanner and use analyze and plot profile to plot the dose profile in ImageJ. Then, mark the irradiation support surface to ensure that the cell container will be placed in the correct position.
To perform a dose rate measurement, place a modified cell container into the irradiation support enclosure and place the ionization chamber into the container in the correct position according to the marks made on the support surface. Confirm that all of the irradiation parameters have been appropriately set and pre-irradiate the ionization chamber for five minutes. Zero the electrometer and obtain 10 one-minute measurements.
Then, use the formula to determine the average dose rate in air kerma. Number all of the films for their downstream identification. At least 24 hours before the irradiation, cut pieces of self-developing dosimetry film according to the size of the cell container.
To construct a calibration curve, set aside three pieces of film for the non-irradiated controls and place the first film inside the cell container. Then, irradiate the film to obtain the first dose point. When all of the films have been irradiated in triplicate at the appropriate selected doses, a calibration curve can be generated.
To measure the cell culture medium attenuation and scattering, select the same irradiation time for all of the irradiations and irradiate three pieces of self-developing dosimetry films in the container without water. Next, place a single piece of film into the container and fill the container with the same volume of water as the volume of medium that will be irradiated. Using small pieces of tape as necessary, position the container within the enclosure.
When the irradiation is complete, dry the films with absorbent paper and store the films protected from light. 24 hours after their irradiation, on the scanner, set the TIFF format to 48-bit red green blue and the transmission mode to 150 dots per inch and select no image correction. To warm up the scanner, place a non-irradiated film on the scanner and launch a preview of the scan.
At the end of the preview, start a timer and wait 30 seconds before starting the scan. At the end of the scan, start a 90-second timer. At the same time, register the scan and open the image in ImageJ.
Trace a square region of interest within the scan and measure the average red pixel level of the area. At the end of the 90 seconds, repeat the scan preview at least 30 times to warm up and stabilize the scanner. Once the scanner has been stabilized, place a film into the center of the scanner bed and launch a preview of the scan.
Wait 30 seconds before starting the scan. At the end of the scan, wait 90 seconds before starting the next scan. Measurements to estimate the attenuator thickness were then performed and the different attenuator thicknesses were tested to find the thickness that decreased the beam intensity by a factor of two.
When this thickness was determined, five measurements were taken to evaluate the average mRAW value corrected by the temperature and pressure correction factor. For this configuration, a half-value layer of 0.667 millimeter of copper was found. In this representative analysis, a self-developing dosimetry film was irradiated to determine the surface on which the irradiation field is homogeneous, allowing correct placement of the cell container.
To determine the exact dose for the cells, the measured air kerma dose rate was converted to water karma using the ratio of the mean mass energy absorption coefficient for water to air evaluated over the photon fluence spectrum, which for this analysis was determined to be 0.659 grays per minute. In this cell culture medium attenuation and scattering analysis, the self-developing dosimetry radiochromic films were first calibrated between zero and three grays with 0.25 gray steps between zero and one gray and 0.5 gray steps between one and three grays. The dose points were then fitted with a fourth-degree polynomial curve.
Here, a representative table used for the daily measurement can be observed. Make sure that all of the configuration parameters are respected, that the does rate is measured inside the right cell container, and that the influence of cell culture medium is evaluated. Several protocols exist to establish a dose reference measurement.
The key point is to select the appropriate protocol for a specific application, especially when using low x-ray facilities.
