Registration procedure for spatial correlation of physical energy deposition of particle irradiation and cellular response utilizing cell-fluorescent ion track hybrid detectors

Abstract

The hybrid technology cell-fluorescent ion track hybrid detector (Cell-Fit-HD) enables the investigation of radiation-related cellular events along single ion tracks on the subcellular scale in clinical ion beams. The Cell-Fit-HD comprises a fluorescent nuclear track detector (FNTD, the physical compartment), a device for individual particle detection and a substrate for viable cell-coating, i.e. the biological compartment. To date both compartments have been imaged sequentially in situ by confocal laser scanning microscopy (CLSM). This is yet in conflict with a functional read-out of the Cell-Fit-HD utilizing a fast live-cell imaging of the biological compartment with low phototoxicity on greater time scales. The read-out of the biological from the physical compartment was uncoupled. A read-out procedure was developed to image the cell layer by conventional widefield microscopy whereas the FNTD was imaged by CLSM. Point mapping registration of the confocal and widefield imaging data was performed. Non-fluorescent crystal defects (spinels) visible in both read-outs were used as control point pairs. The accuracy achieved was on the sub-µm scale. The read-out procedure by widefield microscopy does not impair the unique ability of spatial correlation by the Cell-Fit-HD. The uncoupling will enlarge the application potential of the hybrid technology significantly. The registration allows for an ultimate correlation of microscopic physical beam parameters and cell kinetics on greater time scales. The method reported herein will be instrumental for the introduction of a novel generation of compact detectors facilitating biodosimetric research towards high-throughput analysis.

Carbon ion dosimetry on a fluorescent nuclear track detector using widefield microscopy

“…The registration of track images and cell images captured on the two separate systems introduces a spatial error with regard to the ion track to cell reconstruction. As the images are captured separately on two different occasions and on two different microscopes the field reconstruction is time consuming and involves manually identifying microscopy fields by the inherent colour centre defects in the FNTD [16]. The increased time and effort required to gain an increase in z-axis resolution during FNTD readout along with the fact that the use of two microscopes introduces errors in relation to the captured FOV and the required imaging time strongly diminishes the use of the confocal system [16].…”

“…In contrast, the ideal imaging setup for live cell imaging of cells growing on the FNTD is a widefield microscope (WF) combined with a high sensitivity sCMOS camera as this enables a larger number of frames to be acquired over time and using a tunable LED excitation and a live cell incubation chamber can insure minimal phototoxicity and cellular viability. The current readout process of Cell-FIT-HD for example uses two microscopes to perform in situ bio dosimetry, the imaging routine used to detect the ionizations in an irradiated FNTD makes use of a Zeiss LSM710 confocal microscope [14][15][16]. The second microscope used to image the cells growing on the FNTD was an Olympus IX83 widefield microscope with LED illumination and live cell incubation chamber is used to ensure the cells are growing at a constant 37°C and 5% CO2.…”

“…After the live cell imaging the FNTD is transferred to the LSM where the fluorescent colour centres caused by the traversing ions can be detected. The FNTD images are then retrospectively registered with the initial images taken after irradiation to cross reference the hits to the location of the cells [16]. The readout of the ionization tracks in the FNTD by LSM is currently performed after imaging of the cells growing on the FNTD has been completed.…”

“…The readout of the ionization tracks in the FNTD by LSM is currently performed after imaging of the cells growing on the FNTD has been completed. The imaging of the sample on two microscopes adds spatial uncertainty, on the scale of 1 µm which is rectified by a subsequent image registration of the field of view (FOV) from widefield to confocal [16]. However, the registration of the images between the two microscopes introduces an uncertainty to the ion traversals relative to the cells [16].…”

“…The imaging of the sample on two microscopes adds spatial uncertainty, on the scale of 1 µm which is rectified by a subsequent image registration of the field of view (FOV) from widefield to confocal [16]. However, the registration of the images between the two microscopes introduces an uncertainty to the ion traversals relative to the cells [16]. The readout of one FNTD FOV stack of 20 images (193x193um, 1024x1024px 8-bit image, 40X objective) via LSM takes ~11 minutes using a Zeiss CLSM710.…”