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Technical note: determining regions of interest for CCD camera-based fiber optic luminescence dosimetry by examining signal-to-noise ratio.

ABSTRACT: The goal of this work was to develop a method for determining regions of interest (ROIs) based on signal-to-noise ratio (SNR) for the analysis of charge-coupled device (CCD) images used in luminescence-based radiation dosimetry.The ROI determination method was developed using images containing high-and low-intensity signals taken with a CCD-based, fiber optic plastic scintillation detector system. A series of threshold intensity values was defined for each signal, and ROIs were fitted around the pixels that exceeded each threshold. The SNR for each ROI was calculated and the relationship between SNR and ROI area was examined.The SNR was found to increase rapidly over small ROIs for both signal levels. After reaching a maximum, the SNR of the low-intensity signal decreased steadily over larger ROIs, but the high-intensity SNR did not decrease appreciably over the ROI sizes studied. The spatial extent of the normalized images showed intensity independence, suggesting that a fixed ROI is useful for varying signal levels.The method described here constitutes a simple yet effective method for defining ROIs based on SNR that could enhance the low-level detection capabilities of CCD-based luminescence dosimetry systems.

SUBMITTER: Klein DM

PROVIDER: S-EPMC3055908 | biostudies-other | 2011 Mar

REPOSITORIES: biostudies-other

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Project description:Background:Ca2+ plays an important role in many physiological processes and an accurate study of these signals is important. In modern fluorescence microscopy, a charge-coupled device (CCD) camera is widely deployed for calcium imaging. The ratiometric method is used for the fluorescence dye Fura-2 and Grynkiewitz's formula (Grynkiewicz et al., 1985) is commonly used to convert fluorescence to free Ca2+ concentration ([Ca2+]). But the need to subtract the background signal can lead to a big error in ratiometric calcium measurements. When the error due to background subtraction occurs, the fluorescence ratio of 340 nm divided by 380 nm lights may be twice as large as the actual value. Under conditions when the excitation intensity is not adjusted to ensure the same throughput of the objective lens for ultraviolet dye illumination, the indicator does not gradually bleach out for channels with a wavelength of 340 nm and 380 nm light, which lead to an additional error in determining the concentration of Ca2+. New method:Here we present a new approach for calculating [Ca2+] from the ratiometric fluorescence of Fura-2 dye imaged by a CCD camera. It is designed to optimize [Ca2+] measurements with photobleaching correction without background subtraction error. A mathematical method is also provided for removing the existing underestimated value of fluorescence at an excitation wavelength of 340 nm and compensating for the bleaching rate for both channels with wavelengths of 340 nm and 380 nm using a power function. Results:In cultured neurons, the calculations of the free Ca2+ concentration during Ca2+ transients estimated by the old and new methods, determine it to the same extent. This comparison was made under conditions without errors through background subtraction. If there is this error, the old method calculates [Ca2+] with a much higher, rather than the actual value. Conclusions:We present a modified Grynkiewitz's formula for calculation [Ca2+] for ratiometric dye, such as Fura-2 imaged by a CCD camera, with photobleaching correction without background subtraction error.

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Technical note: determining regions of interest for CCD camera-based fiber optic luminescence dosimetry by examining signal-to-noise ratio.

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