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Investigation of cross talk in single grain luminescence measurements using an EMCCD camera
Radiation Measurements xxx (2015) 1e8
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Investigation of cross talk in single grain luminescence measurements using an EMCCD camera Natacha Gribenski a, *, Frank Preusser a, 1, Steffen Greilich b, Sebastien Huot c, Dirk Mittelstraß d, e a
Department of Physical Geography, Stockholm University, 10691 Stockholm, Sweden Medical Physics in Radiation Oncology, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany Montr D epartement des Sciences de la Terre et de l'atmosph ere, Universit e du Qu ebec a eal, CP 8888 Succ. Centre-Ville, Montr eal, Quebec H3C 3P8, Canada d Freiberg Instruments, Delfter Straße 6, 09599 Freiberg, Germany e Department of Geography, Justus-Liebig-University, Senckenbergstr. 1, 35390 Gießen, Germany b c
h i g h l i g h t s We have performed single grain OSL measurements using an EMCCD detector. Individual equivalent dose cannot be accurately recovered from a mixed dose population. Grains are influenced by signal emitted by their neighbours during the measurements. Simulated data confirm the strong effect of this phenomenon. Increasing the distance between grains or applying brightness criteria are inefficient.
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Article history: Available online xxx
Highly sensitive electron multiplying charges coupled devices (EMCCD) enable the spatial detection of luminescence emissions from samples and have a high potential in single grain luminescence dating. However, the main challenge of this approach is the potential effect of cross talk, i.e. the influence of signal emitted by neighbouring grains, which will bias the information recorded from individual grains. Here, we present the first investigations into this phenomenon when performing single grain luminescence measurements of quartz grains spread over the flat surface of a sample carrier. Dose recovery tests using mixed populations show an important effect of cross talk, even when some distance is kept between grains. This issue is further investigated by focusing just on two grains and complemented by simulated experiments. Creation of an additional rejection criteria based on the brightness properties of the grains is inefficient in selecting grains unaffected by their surroundings. Therefore, the use of physical approaches or image processing algorithms to directly counteract cross talk is essential to allow routine single grain luminescence dating using EMCCD cameras. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Luminescence Spatially resolved EMCCD camera Cross talk Single grain
1. Introduction Conventional optically stimulated luminescence (OSL) dating methods of sediments are based on measuring a signal simultaneously emitted by a multitude of quartz or feldspar minerals. Its successful application will rely on the fullness of exposure to
* Corresponding author. E-mail address: [email protected] (N. Gribenski). 1 Present address: Institute of Earth and Environmental Sciences e Geology, University of Freiburg, Albertstr. 23-B, 79104 Freiburg, Germany.
sunlight, prior deposition. However, when sediments have experienced varying light exposure histories, not necessarily sufficient to completely reset the latent dose, or when post-depositional mixing occurred (e.g., by bioturbation or cryoturbation), it will create an heterogeneous population of mineral grains having accumulated different radiation doses. Analysing the luminescence signal from measurements performed on aliquots containing several grains would lead to the determination of an averaged and hence biased equivalent dose, and so to incorrect age estimates (e.g., Arnold et al., 2012). In these situations, the OSL measurements should be carried out on single grains. The analysis of the
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Please cite this article in press as: Gribenski, N., et al., Investigation of cross talk in single grain luminescence measurements using an EMCCD camera, Radiation Measurements (2015), http://dx.doi.org/10.1016/j.radmeas.2015.01.017
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distribution of single grain equivalent doses allows the detection of partial bleaching or mixing of different doses populations (Olley et al., 1999). Extraction of subpopulations assumed to correspond to the event being dated is then possible with the aid of sophisticated statistical models, such as the minimum age model (Galbraith et al., 1999) or the finite mixture model (Galbraith and Green, 1990). Up until now, routine single grain measurements have relied on a system that employed a focused laser beam moving across a 100 micro-holed disk while sequentially stimulating individual grains in these holes (Duller et al., 1999). However, with the recent technological progress, a new approach using highly-performing imaging detectors, especially electron multiplier charge coupled devices (EMCCD), has been explored repeatedly and promising results relevant to OSL dating have been presented in the past ten years (Greilich et al., 2002; Baril, 2004; Greilich and Wagner, 2006; Clark-Balzan and Schwenninger, 2012). The main advantage of this approach lies in in the broad spectrum of applications that it can offer. In addition to simultaneously measuring the individual signal of a large number of individual grains, such systems could enable OSL measurements of grains in their original context, providing the possibility to consider the effect of microdosimetry (cf. Greilich et al., 2002; Rufer and Preusser, 2009). An EMCCD approach would also allow a larger flexibility in stimulation light sources while assuring a uniform stimulation, which is a potential source of error in the present system (Thomsen et al., 2014). The ultimate aim sought here is to acquire luminescence information from individual grains, randomly spread on a flat surface. Nonetheless, performing routine single grain measurements using EMCCD detectors is hampered by three main issues. Firstly, a welldefined, reproducible and automatic image segmentation method to assign a set of pixels to particular grains is required for individual signal integration. Secondly, it has been observed that the sample carrier can move relatively to the detector over repeated measurements, introducing major uncertainties (Duller et al., 1997; Greilich et al., 2015). Finally, because a grain will emit light in all directions and due to optical aberration, its luminescence signal spreads on the detector over an area larger than its outline. Luminescence signals from adjacent grains could then overlap (cross talk) in the images to be analyzed, contradicting thus the major aim of the approach, i.e. that information is gathered from individual grains. Solutions for the first two issues were recently proposed by Greilich et al. (2015) who developed a specific software, AgesGalore 2, combined with a graphical user interface (AG2-GUI). It includes an automated segmentation approach of the images into discrete areas for signal integration (region of interest, ROI), for each grain. Here, a priori segmentation using reflected light images has been chosen to avoid problems linked to a strong light gradient intensity associated with luminescence images (posteriori segmentation). For each grain, the ROI is defined by following the grain outline as seen in the reflected light images. Furthermore, the issue of relative displacement can easily be monitored and solved by an automated software comparing each reflected light image, performed after each OSL measurement, relatively to the first. The images can then be realigned, as needed, with their associated stack (i.e. the OSL decay curve). However, no investigations into cross talk have been carried out so far. In this article, we present the first observations of image cross talk and its effect on single grain equivalent dose determination using the analytical methods developed by Greilich et al. (2015). This will be accomplished through the aid of dose recovery tests using two grain populations, each given a different artificial dose. The actual spread of light from grains will be quantified and we will explore basic solutions to correct for it.
2. Methodology All measurements were performed using a Princeton Instrument ProEM512B eXcelon EMCCD camera (sensor area of 512 512 pixels, each pixels corresponding to 16 mm 16 mm, with a mapping value of 18 mm per pixel due to optical magnification setup) mounted on a Freiberg Instruments Lexsyg research luminescence reader (Richter et al., 2013). OSL was stimulated using a ring of five LEDs emitting at 458 ± 5 nm with a power of 60 mW cm2 on the sample position. The detection window is limited between 340 nm and 390 nm, by a 2.5 mm Hoya U340 and a Delta BP 365/50 EX interference filter. No colour filters were employed for reflected light images, but a ND40B neutral density filter (ThorLabs, optical density 4.0) and an additional aperture with 8.5 mm inner diameter were used. An internal 90Sr/90Y ring source with a spatial homogeneity >95% over the aliquot area was used (Richter et al., 2012), delivering a dose rate of around 0.05 Gy s1. A ‘solar simulator’ unit consisting of a multi-wavelength LED array was used for bleaching the sample aliquots prior to the experiments. The electron multiplier port of the CCD camera was operated with an avalanche gain of 100 and a readout rate of 5 MHz. Fullchip resolution (512 512 pixels) has been selected in order to preserve a high spatial resolution. By setting an exposure of ~0.5 s per picture, the whole portion of the signal used for analysis is constrained in just one picture, limiting the readout processing events and achieving the highest signal-to-noise ratio. Measurements were performed on quartz grains (160e250 mm, standard chemical pre-treatment) extracted from plunge pool flood sediments from Litchfield National Park, Northern Territories, Australia. According to multiple grains and single grain analyses (performed on a Lexsyg device and a Risø system, respectively), the quartz minerals measured here are especially bright and are dominated by the fast component (May et al., in press). Data analysis was done using the image processing software AG2-GUI (Greilich et al., 2015) that includes an image correction procedure for realignment and removal of extreme high values, caused by bremsstrahlung events originating from cosmic rays and the nearby beta sources (two at ca. 20 cm). The software also analyses individual signals integrated over the areas defined by the grain boundaries. Equivalent doses and their associated error were calculated using an exponential curve fitting and the ‘curve fitting’ uncertainty approximation described in Duller (2007). Grains for which the signal was not significant above background or where its equivalent dose was larger than 2D0 have been rejected (Wintle and Murray, 2006). The Single Aliquot Regenerative Dose (SAR) protocol, with a preheat step of 10 s at 230 C and a blue LED OSL stimulation for 30 s at 125 C, has been used for all dose recovery experiments (Table 1). The net signal consists of the first picture (the initial 0.5 s of stimulation) minus a background, taken from the average values in pictures 7 to 9 (3.0e4.5 s). The reliability of this protocol was previously tested for this material on a population having received
Table 1 Dose recovery test protocol. Dose induced Preheat 230 C for 10 s OSL measurements 30 s at 125 C Reflected light photo Sunlight simulator bleaching 150 s, 50% power Test Dose (5 Gy) Preheat 230 C for 10 s OSL measurements 30 s at 125 C Reflected light photo Sunlight simulator bleaching 150 s, 50% power
Please cite this article in press as: Gribenski, N., et al., Investigation of cross talk in single grain luminescence measurements using an EMCCD camera, Radiation Measurements (2015), http://dx.doi.org/10.1016/j.radmeas.2015.01.017
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a uniform dose of 50 Gy, after bleaching. 110 grains showing low recuperation (<0.15% of natural) and recycling ratios close to unity (0.8e1.2) were successfully measured with the EMCCD. Using the central age model (CAM) to calculate the weighted mean, we obtained a recovered to given ratio of 1.038 ± 0.015 (51.9 ± 0.75 Gy, with an overdispersion of 13%) (Greilich et al., 2015). 3. Investigating the effect of cross talk 3.1. Dose recovery test on a mixed population sample In order to obtain a first estimate of the potential effect of cross talk, a dose recovery test was performed on a sample comprising of a mix of two populations of grains that had previously been bleached and given an artificial dose of ca. 10 Gy and or ca. 50 Gy, respectively. Around 250 grains were spread over the surface of two stainless steel disks, taking care that they were well separated by checking the disk under a stereomicroscope and using a needle when necessary (Fig. 1a). A total of 104 single grain equivalent doses were recovered. Their measured dose distribution exhibited a wide dispersion between 10 Gy and 50 Gy. The overdispersion is similar, at 34%, whether we considered all the grains (Fig. 1b) or retained only those having a low recuperation ratio (<0.1) and a recycling ratio close to unity (0.8e1.2; Fig. 1c). The lack of any significant peak distribution around the given doses of 10 and 50 Gy is reminiscent of Arnold et al. (2012) pseudo grains prediction. Given that the previous dose recovery, on a single dose population (cf. section 2.), was successful, it would appear here that the luminescence intensity from any given grain is influenced by its neighbours, i.e. that cross talk has a major impact on dose recovery. 3.2. Assessing of light spread from a grain In this experiment we investigate how far the OSL signal laterally spreads beyond the grain boundaries defined by AG2-GUI, on basis of reflected light pictures. Fig. 2 shows various normalized light intensity profiles extracted from the OSL measurement images of an especially bright grain previously irradiated with 100, 50, 20, 10 and 5 Gy. The overall shape is more or less similar, with the bulk intensity concentrated within the physical grain boundary. However, it does spread further. The length of grain ROIs defined by AG2-GUI for this sample is around 270e360 mm (15e20 pixels). Of the total signal recorded over 2350 mm, more than 60% is located directly above the grain ROI (i.e. ca. 325 mm length) and around 80%
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is found within a distance of 450 mm on each sides from the center of the grain (around 300 mm beyond the grain boundary). On this same distance, we observe a dramatic decrease of the signal (to less than 20% of the maximal intensity), which continues to decrease beyond, reaching intensities below 5% (on average). This implies that maintaining every grain at a distance of around 360 mm (20 pixels) from each other (corresponding to more or less the maximum length of a grain ROI) should limit the cross talk from adjacent grains. Besides, it would also allow to dispense a sizeable amount of grains over the disk (>100), making it worthwhile for routine application.
3.3. Cross talk influence of one grain on another Experiments carried out on a large number of grains spread on the disk will not allow for a specific observation of how cross talk affects the recorded signal from individual grains. To simplify the problem and for a better understanding of this phenomenon we limited the test to just two grains (A and B). These grains were selected among several hundred grains for their similar brightness and good behaviour to the standard SAR tests (dose recovery ratio within 5% of unity and test dose sensitivity change inferior to 10%). Repeated dose recovery tests were performed, for which the reference grain (A) always received the same dose (10 Gy) while increasing doses (20, 50 and 100 Gy) were given to grain B, by this acting as the influencing agent. For each experiment, a distance of around 360 mm (20 pixels) was kept between the two grains. The same protocol as before was applied, but with lower artificial doses induced (ca. 5, 10, 15 Gy; test dose ca. 5 Gy). We observed an important offset in the recovered doses from the reference grain (A). For a given dose of 20 and 50 Gy to grain B, we obtained recovery ratios of 1.17 ± 0.02% and 1.20 ± 0.03%, respectively, which went up to 1.41 ± 0.03% for a given dose of 100 Gy to grain B. However, these results could be biased by several factors due to the setting of the experiment. For instance, although we paid attention, keeping the exactly same distance between grains during aliquot preparation is not possible. The same applies to the orientation and to the actual location of the grain on the disk, which could have an effect during the irradiation and measurement steps. Finally, new ROIs need to be defined for each experiment and this could also add to our uncertainties. Nevertheless, all these sources of uncertainties cannot explain why the given dose is systematically overestimated and presumably this is due to the effect of cross talk.
Fig. 1. The reflected light image (a) showing an example of the disposition of grains on a disc (about 130 grains) used for the experiment reported here. The graphs show the distribution of measured equivalent doses (b, n ¼ 104) without any rejection criteria applied and (c, n ¼ 27) with rejection of grains for which the recuperation ratio was above 0.1 or the recycling ratios outside 20% of unity. No peaks centered at 10 Gy and 50 Gy are distinguishable.
Please cite this article in press as: Gribenski, N., et al., Investigation of cross talk in single grain luminescence measurements using an EMCCD camera, Radiation Measurements (2015), http://dx.doi.org/10.1016/j.radmeas.2015.01.017
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Fig. 2. Normalized light intensities profiles extracted from the OSL measurement images of an especially bright grain previously irradiated with 100, 50, 20, 10 and 5 Gy. The length of grain ROIs defined by AG2-GUI for this sample is around 270e360 mm. Signal is given in analog to digital units (a.d.u.). Around 80% of the signal is concentrated within a distance of 450 mm on each sides from the center of the grain.
3.4. Simulation of the cross talk effect To overcome the issues discussed above, and to ensure that the results strictly reflect the cross talk effect, we created a small mathematical model in Matlab. The mathematical reconstruct relied on actual OSL measurements that were recorded in the case of a unique, well behaving grain, present on the disk. Luminescence emissions from two identical grains were simulated at varying distances and with varying doses given. Dose recovery tests were then
reconstituted with, as previously, one reference grain receiving a constant dose of 10 Gy, and the influencing grain receiving an increasing initial dose (20, 50 and 100 Gy) and being located at varying distances (180, 360, 720 and 1080 mm). A fix ROI of 324 mm (18 pixels) of diameter was applied to integrate the signal of the reference grain. This approach allows us to eliminate the potential sources of errors pointed out during the real experiment (cf. section 3.2.). In total, twelve scenarios have been generated to observe the influence of one grain on another (Fig. 3.). We run an additional
Fig. 3. Simulation of 12 scenarios of dose recovery tests, for varying given dose to the influencing grain (grain on the right hand-side) and varying distances between the two grains. The simulations are based on real measurements originating from one grain selected for its brightness and good response to standard SAR protocol. Recovery ratios are indicated for the reference grain (on the left hand-side).
Please cite this article in press as: Gribenski, N., et al., Investigation of cross talk in single grain luminescence measurements using an EMCCD camera, Radiation Measurements (2015), http://dx.doi.org/10.1016/j.radmeas.2015.01.017
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simulation for a single, unique grain present on the disk, for comparison, which provided consistent results (recovery ratio of 1.03 ± 0.02 and test dose sensitivity change inferior to 5%). As expected, under the influence of another grain, the offset of the recovered doses of the reference grain (A) increases as the dose induced to the grain B increases and as the distance between the two grains reduces. When compared to the results obtained from the real measurements and for similar conditions (distance of 360 mm and doses of 20, 50 and 100 Gy given to grain B), we can note that these simulations yielded similar or higher offsets (1.17 ± 0.02, 1.20 ± 0.02 and 1.41 ± 0.03 versus 1.13 ± 0.02, 1.32 ± 0.02 and 1.46 ± 0.02, respectively). Thus, in all likelihood, cross talk from a nearby grain is the dominant factor here that hampers our ability to accurately measure a dose in grain A. 3.5. Evaluation of the cross talk effect During measurements based on a traditional SAR protocol, influence from surrounding grains will affect the determination of individual equivalent dose in two ways: in the natural measurement and for every laboratory-induced doses used to construct the growth curve. Based on our simulation results with two grains of identical brightness properties, we observe that the effect of an adjacent grain on the original growth curve is relatively limited, as the curve is only slightly displaced (Fig. 4). Thus, for a constant Lx/Tx ratio, it would yield to a maximum variation of the inferred equivalent dose of 0.82 Gy (Lx/Tx corresponding to an equivalent dose around ca. 20 Gy; 0.33 Gy of variation for Lx/Tx ca. 10 Gy) for various distance between the grains. The fact that the Lx/Tx varies little bears resemblance to the fact that laboratory-induced luminescence in quartz is relatively universal and standard in shape (Roberts and Duller, 2004). We record an increasing of the original equivalent dose of up to 7 Gy in our simulations (scenario with smallest distance between grains and maximum dose given to the influencing grain). Therefore, the main source of error is due to the
Fig. 5. Proportion by which the “natural” signal of the reference grain is increased, for different scenarios of initial dose given to the influencing grain and distance between them.
additional signal coming from the surrounding grain during the ‘natural signal’ measurement. In our simulation, this translates into an increasing of the original signal from 10% to more than 65% (Fig. 5), yielding to a variation of the initial Ln/Tn ratio from 1.87 to up to 2.70. However, these statements apply only for the very specific greatly controlled situation in this experiment (i.e. influence from only one grain, identical brightness properties, distance controlled). It is likely that the influence of cross talk on growth curve construction and especially initial signal measurements greatly increases for a more realistic situation when many grains are randomly spread on the aliquot with varying brightness properties and original doses (in the case of a partially bleached sediment). Finally, the simulations show that even keeping a much greater distance between grains (1080 mm) than the safety distance
Fig. 4. Influence of cross talk on the growth curve construction of the reference grain, for different distances with the influencing grain. The red curve represents the growth curve for a grain sitting all alone. The effect on the resulting equivalent dose is limited with a maximum variation of 0.82 Gy (upper right, Lx/Tx corresponding to ~20 Gy), and a variation of maximum 0.33 Gy in the area of the curve for a dose corresponding to around 10 Gy. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: Gribenski, N., et al., Investigation of cross talk in single grain luminescence measurements using an EMCCD camera, Radiation Measurements (2015), http://dx.doi.org/10.1016/j.radmeas.2015.01.017
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previously suggested (360 mm, section 3.2.) is insufficient to avoid a significant cross talk effect, in case of large difference of initial doses and brightness properties between the grains.
4. Potential solutions to counteract the effect of cross talk The experiments above have shown that performing single grain measurements with an EMCCD detector and using the AG2GUI advised method of image processing at its current stage of development does not allow obtaining reliable individual signal and equivalent dose estimates per grain. Additional procedures are therefore necessary to overcome the effect of cross talk influence from adjacent grains. Fig. 7. Recovery ratio (filled circles) of the reference grain (A) in relation to the dose given to the neighbouring grain (B). The results were improved with the use of the Background subtractor plugin (open circles).
4.1. Brightness criteria As a first possibility, one could try to apply an additional criteria based on the brightness of the grains, assuming that the brightest grains should be less sensitive to luminescence influence from surrounding grains. To test if such criteria could be relevant, we plotted the net ‘natural signals’ (averaged through ROI) of the single grains obtained from the first experiment (cf. section 3.1.) against their equivalent dose (Fig. 6a). The same was performed, but by using the net signals obtained for a 60 Gy regeneration dose (Fig. 6b). By this way, the signal can be approximated as a luminescence index since every grain received the same induced dose. In both cases, the majority of the data points are concentrated within a limited range of signal strength (from 25 to 100, for Fig. 6a and from 50 to 125, for Fig. 6b, analog-to-digital units) and the measured single grain doses are widely scattered, without any correlation between signal strength and the correct dose value recovered (10 or 50 Gy). For the data points exhibiting outstanding brightness, very few grains recover, indeed, the initial dose given. However, other grains with similar or slightly lower brightness properties are still widely spread between 10 Gy and 50 Gy. Therefore, these results show that discriminating grains based on their brightness properties to avoid single grain doses biased by cross talk is not efficient. In addition, applying such a procedure would lead to the exclusion of an important number of grains, thus reducing the appeal of an EMCCD approach. Hence, seeking corrective procedure to remove the effect of cross talk in every grain is preferable.
4.2. Potential of image processing algorithms: example of the “Background Subtractor” tool One potential solution is the use of image processing algorithms. Numerous algorithms, from simple to very complex, are widely used and constantly developed for processing images and analyzing light intensities of various objects in many different fields (such as micro-biology, medical sciences and photometry astronomy). For our purpose, we tested the potential of such approaches by using the Background Subtractor plugin software for ImageJ (Cardinale, 2010), which is based on a local histogram approach through a sliding square window (20 pixels here) to calculate and remove the local inconstant background illumination. By applying this algorithm on the luminescence images, we aim at reducing the blurring of the luminescent objects and so the cross talk effect, before integrating the signal and calculating the equivalent doses. First, we applied this additional step to the original image data sets obtained during the one-to-one grain real experiment (cf. section 3.3.). When compared to the initial recovered doses of the reference grain, we observe a clear improvement, with very satisfying recovery ratios (<10% offset) when the neighbouring grain received initial doses of 20 and 50 Gy (Fig. 7). The dose recovery test only still failed (recovery ratio of 1.22 ± 0.05) when a dose of 100 Gy is given to the neighbouring grain. The use of the Background Subtractor plugin to reduce the effects induced by cross talk seems relatively successful in that situation.
Fig. 6. The net natural signal (a) and the net signal of a regenerative dose of 60 Gy (b), averaged through the ROIs, are plotted against equivalent doses. Signal is given in analog to digital units (a.d.u.). No clear trend between brightness strength and recovered equivalent dose is observed.
Please cite this article in press as: Gribenski, N., et al., Investigation of cross talk in single grain luminescence measurements using an EMCCD camera, Radiation Measurements (2015), http://dx.doi.org/10.1016/j.radmeas.2015.01.017
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Fig. 8. Distribution of single grain equivalent doses obtained when applying an additional step of local background removal using the Background Substractor plugin on the luminescence images. (a) Distribution without any rejection criteria (n ¼ 124); (b) equivalent dose distribution when grains with recuperation ratios >0.1 or recycling ratios outside 20% of unity are rejected (n ¼ 47). In both cases, no peaks at 10 and 50 Gy are distinguishable.
However, it was necessary to test how it would perform in more realistic circumstances, when there is a multitude of grains of varying brightness separated by varying distances on the aliquot surface. For this, the plugin was applied to the image data set obtained during the first dose recovery test performed on aliquots with more than one hundred grains spread on the surface (cf. section 3.1.). The distribution of the single grain equivalent doses obtained is still widely dispersed, for both when all grains are considered (Fig. 8a), or when rejection criteria are applied (recuperation ratio >0.1, recycling ratio outside 0.8 and 1.2; Fig. 8b). No distinct grouping around 10 Gy and 50 Gy are observed, as it would be expected in the case of success. However, we note that the number of grains for which an equivalent dose could be calculated slightly increased (124 versus 104 grains, and 45 versus 27, respectively), and the uncertainties are reduced (relative error around 20e30 % in average instead of 40e70 %). The Background subtractor removes local background intensities by evaluating the frequency of pixel intensities for a given surrounding. It can be assumed that part of this background is given by the relatively flat cross talk signal. Hence, application of the plugin reduces the portion of additional signal coming from surrounding grains and reduces the uncertainty and the spreading of the normalized data points used for the growth curve reconstruction.
5. Discussion and conclusions The experiments carried out in this study show that grains spread over a flat surface without further precaution are clearly influenced by signal emitted by surrounding grains, when performing single grain EMCCD luminescent measurements. However, it is very difficult to quantitatively assess the effect of cross talk in general, as it will probably highly vary according to the samples being measured. Indeed, the influence of this phenomenon will be strongly modulated by the gradient intensity between grains, inherent to their brightness properties and/or to their accumulated dose. Besides, the effect will be amplified as each grain will be influenced by a cumulated signal emitted by numerous surrounding grains. Experiments showed that keeping a ‘safety’ distance between grains is insufficient in minimizing cross talk effect and counteracts the desire to have a sizeable amount of grains over the disk in order to maximize the statistical output of one aliquot. Introducing
brightness criteria to reject single grain doses more susceptible to be significantly biased by the phenomenon also proved to be inadequate. With regard to these elements, we consider the successful measurements of single grains spread on a flat surface while using an EMCCD camera cannot be performed without introducing an additional action to directly counteract or correct for the cross talk influence from surrounding grains. For this, several potential solution exist. The most straightforward is to set a physical barrier between grains by using a special sample carrier such as used by the Risø system (Thomsen et al., 2014). Although it probably will not totally prevent cross talk, as the effect is not only physically in nature, due to a lateral spreading of light once it comes of the grain, but also due to optical effects, such as blurring in the lens. In addition, the use of a disk with a fixed geometry (e.g. drilled holes) would negate the possibility to randomly spread grains on a flat surface, allowing, for example, the use of different grains sizes. Further development in the optical setup might as well reduce blurring. However, this would certainly be at the cost of a loss in sensitivity and in detection window flexibility. Finally, we showed that the results obtained in the simple situation when using only two grains with moderate brightness difference can greatly be improved by applying a relatively basic imaging processing tool. Therefore, we strongly encourage further exploration of image processing solutions based on more sophisticated algorithms. In case of success, this approach would open much more possibilities to use EMCCD cameras in luminescence dating, beyond the traditional measurement of well separated grains.
Acknowledgements We are grateful for very constructive reviewer's comments that helped us to improve the presentation of this study. We thank Krister Jansson for his valuable suggestions on how to improve the flow of the text, as well as for his continuous support and that of Arjen Stroeven and Jonathan Harbor. We also acknowledge Irina Rogozhina and Lucile Verrot for their patient advising in computer programming. This work was supported by a Swedish research Council grant to Arjen P. Stroeven and Krister N. Jansson (No. 20114892 and 2009-4411, respectively), and Sebastien Huot received funding from the CFI-FEI (Canada Foundation for Innovation) during this work.
Please cite this article in press as: Gribenski, N., et al., Investigation of cross talk in single grain luminescence measurements using an EMCCD camera, Radiation Measurements (2015), http://dx.doi.org/10.1016/j.radmeas.2015.01.017
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luminescence: a unique tool for archaeochronometry. Naturwissenschaften 89, 371e375. Greilich S., Gribenski N., Mittelstrass D., Dornich K.,Huot S., Preusser F., Single grain dose distribution measurement by optically stimulated luminescence using an integrated EMCCD-based system, arXive 2015, arXive:1502.01204. Available on: http://arxiv.org/abs/1502.01204. May J.-H., Preusser F., Gliganic L.A. Refining late Quaternary plunge pool chronologies in Australia's monsoonal ‘Top End’. Quat. Geochronol. doi:10.1016/j.quageo.2015.01.008, (in press) Olley, J.M., Caitcheon, G.G., Roberts, R.G., 1999. The origin of dose distributions in fluvial sediments, and the prospect of dating single grains from fluvial deposits using optically stimulated luminescence. Radiat. Meas. 30, 207e217. Richter, D., Pintaske, R., Dornich, K., Krbetscheck, M., 2012. A novel beta source design for uniform irradiation in dosimetric applications. Anc. TL 30, 57e64. Richter, D., Richter, A., Dornich, K., 2013. Lexsyg - a new system for luminescence research. Geochronometria 40, 220e228. Roberts, H.M., Duller, G.A.T., 2004. Standardised growth curves for optical dating of sediment using multiple-grain aliquots. Radiat. Meas. 38, 241e252. Rufer, D., Preusser, F., 2009. Potential of autoradiography to detect spatially resolved radiation patterns in the context of trapped charge dating. Geochronometria 34, 1e13. Thomsen, K.J., Kook, M.H., Murray, A.S., Jain, M., Lapp, T., 2014. Single grain dose estimations form an EMCCD-based imaging system. In: Abstract Volume of the 14th International Conference on Luminescence and Electron Spin Resonance al, Canada. Dating. July 7e11. Montre Wintle, A.G., Murray, A.S., 2006. A review of quartz optically stimulated luminescence characteristics and their relevance in single-aliquot regeneration dating protocols. Radiat. Meas. 41, 369e391.
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Radiation Measurements journal homepage: www.elsevier.com/locate/radmeas
Investigation of cross talk in single grain luminescence measurements using an EMCCD camera Natacha Gribenski a, *, Frank Preusser a, 1, Steffen Greilich b, Sebastien Huot c, Dirk Mittelstraß d, e a
Department of Physical Geography, Stockholm University, 10691 Stockholm, Sweden Medical Physics in Radiation Oncology, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany Montr D epartement des Sciences de la Terre et de l'atmosph ere, Universit e du Qu ebec a eal, CP 8888 Succ. Centre-Ville, Montr eal, Quebec H3C 3P8, Canada d Freiberg Instruments, Delfter Straße 6, 09599 Freiberg, Germany e Department of Geography, Justus-Liebig-University, Senckenbergstr. 1, 35390 Gießen, Germany b c
h i g h l i g h t s We have performed single grain OSL measurements using an EMCCD detector. Individual equivalent dose cannot be accurately recovered from a mixed dose population. Grains are influenced by signal emitted by their neighbours during the measurements. Simulated data confirm the strong effect of this phenomenon. Increasing the distance between grains or applying brightness criteria are inefficient.
a r t i c l e i n f o
a b s t r a c t
Article history: Available online xxx
Highly sensitive electron multiplying charges coupled devices (EMCCD) enable the spatial detection of luminescence emissions from samples and have a high potential in single grain luminescence dating. However, the main challenge of this approach is the potential effect of cross talk, i.e. the influence of signal emitted by neighbouring grains, which will bias the information recorded from individual grains. Here, we present the first investigations into this phenomenon when performing single grain luminescence measurements of quartz grains spread over the flat surface of a sample carrier. Dose recovery tests using mixed populations show an important effect of cross talk, even when some distance is kept between grains. This issue is further investigated by focusing just on two grains and complemented by simulated experiments. Creation of an additional rejection criteria based on the brightness properties of the grains is inefficient in selecting grains unaffected by their surroundings. Therefore, the use of physical approaches or image processing algorithms to directly counteract cross talk is essential to allow routine single grain luminescence dating using EMCCD cameras. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Luminescence Spatially resolved EMCCD camera Cross talk Single grain
1. Introduction Conventional optically stimulated luminescence (OSL) dating methods of sediments are based on measuring a signal simultaneously emitted by a multitude of quartz or feldspar minerals. Its successful application will rely on the fullness of exposure to
* Corresponding author. E-mail address: [email protected] (N. Gribenski). 1 Present address: Institute of Earth and Environmental Sciences e Geology, University of Freiburg, Albertstr. 23-B, 79104 Freiburg, Germany.
sunlight, prior deposition. However, when sediments have experienced varying light exposure histories, not necessarily sufficient to completely reset the latent dose, or when post-depositional mixing occurred (e.g., by bioturbation or cryoturbation), it will create an heterogeneous population of mineral grains having accumulated different radiation doses. Analysing the luminescence signal from measurements performed on aliquots containing several grains would lead to the determination of an averaged and hence biased equivalent dose, and so to incorrect age estimates (e.g., Arnold et al., 2012). In these situations, the OSL measurements should be carried out on single grains. The analysis of the
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distribution of single grain equivalent doses allows the detection of partial bleaching or mixing of different doses populations (Olley et al., 1999). Extraction of subpopulations assumed to correspond to the event being dated is then possible with the aid of sophisticated statistical models, such as the minimum age model (Galbraith et al., 1999) or the finite mixture model (Galbraith and Green, 1990). Up until now, routine single grain measurements have relied on a system that employed a focused laser beam moving across a 100 micro-holed disk while sequentially stimulating individual grains in these holes (Duller et al., 1999). However, with the recent technological progress, a new approach using highly-performing imaging detectors, especially electron multiplier charge coupled devices (EMCCD), has been explored repeatedly and promising results relevant to OSL dating have been presented in the past ten years (Greilich et al., 2002; Baril, 2004; Greilich and Wagner, 2006; Clark-Balzan and Schwenninger, 2012). The main advantage of this approach lies in in the broad spectrum of applications that it can offer. In addition to simultaneously measuring the individual signal of a large number of individual grains, such systems could enable OSL measurements of grains in their original context, providing the possibility to consider the effect of microdosimetry (cf. Greilich et al., 2002; Rufer and Preusser, 2009). An EMCCD approach would also allow a larger flexibility in stimulation light sources while assuring a uniform stimulation, which is a potential source of error in the present system (Thomsen et al., 2014). The ultimate aim sought here is to acquire luminescence information from individual grains, randomly spread on a flat surface. Nonetheless, performing routine single grain measurements using EMCCD detectors is hampered by three main issues. Firstly, a welldefined, reproducible and automatic image segmentation method to assign a set of pixels to particular grains is required for individual signal integration. Secondly, it has been observed that the sample carrier can move relatively to the detector over repeated measurements, introducing major uncertainties (Duller et al., 1997; Greilich et al., 2015). Finally, because a grain will emit light in all directions and due to optical aberration, its luminescence signal spreads on the detector over an area larger than its outline. Luminescence signals from adjacent grains could then overlap (cross talk) in the images to be analyzed, contradicting thus the major aim of the approach, i.e. that information is gathered from individual grains. Solutions for the first two issues were recently proposed by Greilich et al. (2015) who developed a specific software, AgesGalore 2, combined with a graphical user interface (AG2-GUI). It includes an automated segmentation approach of the images into discrete areas for signal integration (region of interest, ROI), for each grain. Here, a priori segmentation using reflected light images has been chosen to avoid problems linked to a strong light gradient intensity associated with luminescence images (posteriori segmentation). For each grain, the ROI is defined by following the grain outline as seen in the reflected light images. Furthermore, the issue of relative displacement can easily be monitored and solved by an automated software comparing each reflected light image, performed after each OSL measurement, relatively to the first. The images can then be realigned, as needed, with their associated stack (i.e. the OSL decay curve). However, no investigations into cross talk have been carried out so far. In this article, we present the first observations of image cross talk and its effect on single grain equivalent dose determination using the analytical methods developed by Greilich et al. (2015). This will be accomplished through the aid of dose recovery tests using two grain populations, each given a different artificial dose. The actual spread of light from grains will be quantified and we will explore basic solutions to correct for it.
2. Methodology All measurements were performed using a Princeton Instrument ProEM512B eXcelon EMCCD camera (sensor area of 512 512 pixels, each pixels corresponding to 16 mm 16 mm, with a mapping value of 18 mm per pixel due to optical magnification setup) mounted on a Freiberg Instruments Lexsyg research luminescence reader (Richter et al., 2013). OSL was stimulated using a ring of five LEDs emitting at 458 ± 5 nm with a power of 60 mW cm2 on the sample position. The detection window is limited between 340 nm and 390 nm, by a 2.5 mm Hoya U340 and a Delta BP 365/50 EX interference filter. No colour filters were employed for reflected light images, but a ND40B neutral density filter (ThorLabs, optical density 4.0) and an additional aperture with 8.5 mm inner diameter were used. An internal 90Sr/90Y ring source with a spatial homogeneity >95% over the aliquot area was used (Richter et al., 2012), delivering a dose rate of around 0.05 Gy s1. A ‘solar simulator’ unit consisting of a multi-wavelength LED array was used for bleaching the sample aliquots prior to the experiments. The electron multiplier port of the CCD camera was operated with an avalanche gain of 100 and a readout rate of 5 MHz. Fullchip resolution (512 512 pixels) has been selected in order to preserve a high spatial resolution. By setting an exposure of ~0.5 s per picture, the whole portion of the signal used for analysis is constrained in just one picture, limiting the readout processing events and achieving the highest signal-to-noise ratio. Measurements were performed on quartz grains (160e250 mm, standard chemical pre-treatment) extracted from plunge pool flood sediments from Litchfield National Park, Northern Territories, Australia. According to multiple grains and single grain analyses (performed on a Lexsyg device and a Risø system, respectively), the quartz minerals measured here are especially bright and are dominated by the fast component (May et al., in press). Data analysis was done using the image processing software AG2-GUI (Greilich et al., 2015) that includes an image correction procedure for realignment and removal of extreme high values, caused by bremsstrahlung events originating from cosmic rays and the nearby beta sources (two at ca. 20 cm). The software also analyses individual signals integrated over the areas defined by the grain boundaries. Equivalent doses and their associated error were calculated using an exponential curve fitting and the ‘curve fitting’ uncertainty approximation described in Duller (2007). Grains for which the signal was not significant above background or where its equivalent dose was larger than 2D0 have been rejected (Wintle and Murray, 2006). The Single Aliquot Regenerative Dose (SAR) protocol, with a preheat step of 10 s at 230 C and a blue LED OSL stimulation for 30 s at 125 C, has been used for all dose recovery experiments (Table 1). The net signal consists of the first picture (the initial 0.5 s of stimulation) minus a background, taken from the average values in pictures 7 to 9 (3.0e4.5 s). The reliability of this protocol was previously tested for this material on a population having received
Table 1 Dose recovery test protocol. Dose induced Preheat 230 C for 10 s OSL measurements 30 s at 125 C Reflected light photo Sunlight simulator bleaching 150 s, 50% power Test Dose (5 Gy) Preheat 230 C for 10 s OSL measurements 30 s at 125 C Reflected light photo Sunlight simulator bleaching 150 s, 50% power
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a uniform dose of 50 Gy, after bleaching. 110 grains showing low recuperation (<0.15% of natural) and recycling ratios close to unity (0.8e1.2) were successfully measured with the EMCCD. Using the central age model (CAM) to calculate the weighted mean, we obtained a recovered to given ratio of 1.038 ± 0.015 (51.9 ± 0.75 Gy, with an overdispersion of 13%) (Greilich et al., 2015). 3. Investigating the effect of cross talk 3.1. Dose recovery test on a mixed population sample In order to obtain a first estimate of the potential effect of cross talk, a dose recovery test was performed on a sample comprising of a mix of two populations of grains that had previously been bleached and given an artificial dose of ca. 10 Gy and or ca. 50 Gy, respectively. Around 250 grains were spread over the surface of two stainless steel disks, taking care that they were well separated by checking the disk under a stereomicroscope and using a needle when necessary (Fig. 1a). A total of 104 single grain equivalent doses were recovered. Their measured dose distribution exhibited a wide dispersion between 10 Gy and 50 Gy. The overdispersion is similar, at 34%, whether we considered all the grains (Fig. 1b) or retained only those having a low recuperation ratio (<0.1) and a recycling ratio close to unity (0.8e1.2; Fig. 1c). The lack of any significant peak distribution around the given doses of 10 and 50 Gy is reminiscent of Arnold et al. (2012) pseudo grains prediction. Given that the previous dose recovery, on a single dose population (cf. section 2.), was successful, it would appear here that the luminescence intensity from any given grain is influenced by its neighbours, i.e. that cross talk has a major impact on dose recovery. 3.2. Assessing of light spread from a grain In this experiment we investigate how far the OSL signal laterally spreads beyond the grain boundaries defined by AG2-GUI, on basis of reflected light pictures. Fig. 2 shows various normalized light intensity profiles extracted from the OSL measurement images of an especially bright grain previously irradiated with 100, 50, 20, 10 and 5 Gy. The overall shape is more or less similar, with the bulk intensity concentrated within the physical grain boundary. However, it does spread further. The length of grain ROIs defined by AG2-GUI for this sample is around 270e360 mm (15e20 pixels). Of the total signal recorded over 2350 mm, more than 60% is located directly above the grain ROI (i.e. ca. 325 mm length) and around 80%
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is found within a distance of 450 mm on each sides from the center of the grain (around 300 mm beyond the grain boundary). On this same distance, we observe a dramatic decrease of the signal (to less than 20% of the maximal intensity), which continues to decrease beyond, reaching intensities below 5% (on average). This implies that maintaining every grain at a distance of around 360 mm (20 pixels) from each other (corresponding to more or less the maximum length of a grain ROI) should limit the cross talk from adjacent grains. Besides, it would also allow to dispense a sizeable amount of grains over the disk (>100), making it worthwhile for routine application.
3.3. Cross talk influence of one grain on another Experiments carried out on a large number of grains spread on the disk will not allow for a specific observation of how cross talk affects the recorded signal from individual grains. To simplify the problem and for a better understanding of this phenomenon we limited the test to just two grains (A and B). These grains were selected among several hundred grains for their similar brightness and good behaviour to the standard SAR tests (dose recovery ratio within 5% of unity and test dose sensitivity change inferior to 10%). Repeated dose recovery tests were performed, for which the reference grain (A) always received the same dose (10 Gy) while increasing doses (20, 50 and 100 Gy) were given to grain B, by this acting as the influencing agent. For each experiment, a distance of around 360 mm (20 pixels) was kept between the two grains. The same protocol as before was applied, but with lower artificial doses induced (ca. 5, 10, 15 Gy; test dose ca. 5 Gy). We observed an important offset in the recovered doses from the reference grain (A). For a given dose of 20 and 50 Gy to grain B, we obtained recovery ratios of 1.17 ± 0.02% and 1.20 ± 0.03%, respectively, which went up to 1.41 ± 0.03% for a given dose of 100 Gy to grain B. However, these results could be biased by several factors due to the setting of the experiment. For instance, although we paid attention, keeping the exactly same distance between grains during aliquot preparation is not possible. The same applies to the orientation and to the actual location of the grain on the disk, which could have an effect during the irradiation and measurement steps. Finally, new ROIs need to be defined for each experiment and this could also add to our uncertainties. Nevertheless, all these sources of uncertainties cannot explain why the given dose is systematically overestimated and presumably this is due to the effect of cross talk.
Fig. 1. The reflected light image (a) showing an example of the disposition of grains on a disc (about 130 grains) used for the experiment reported here. The graphs show the distribution of measured equivalent doses (b, n ¼ 104) without any rejection criteria applied and (c, n ¼ 27) with rejection of grains for which the recuperation ratio was above 0.1 or the recycling ratios outside 20% of unity. No peaks centered at 10 Gy and 50 Gy are distinguishable.
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Fig. 2. Normalized light intensities profiles extracted from the OSL measurement images of an especially bright grain previously irradiated with 100, 50, 20, 10 and 5 Gy. The length of grain ROIs defined by AG2-GUI for this sample is around 270e360 mm. Signal is given in analog to digital units (a.d.u.). Around 80% of the signal is concentrated within a distance of 450 mm on each sides from the center of the grain.
3.4. Simulation of the cross talk effect To overcome the issues discussed above, and to ensure that the results strictly reflect the cross talk effect, we created a small mathematical model in Matlab. The mathematical reconstruct relied on actual OSL measurements that were recorded in the case of a unique, well behaving grain, present on the disk. Luminescence emissions from two identical grains were simulated at varying distances and with varying doses given. Dose recovery tests were then
reconstituted with, as previously, one reference grain receiving a constant dose of 10 Gy, and the influencing grain receiving an increasing initial dose (20, 50 and 100 Gy) and being located at varying distances (180, 360, 720 and 1080 mm). A fix ROI of 324 mm (18 pixels) of diameter was applied to integrate the signal of the reference grain. This approach allows us to eliminate the potential sources of errors pointed out during the real experiment (cf. section 3.2.). In total, twelve scenarios have been generated to observe the influence of one grain on another (Fig. 3.). We run an additional
Fig. 3. Simulation of 12 scenarios of dose recovery tests, for varying given dose to the influencing grain (grain on the right hand-side) and varying distances between the two grains. The simulations are based on real measurements originating from one grain selected for its brightness and good response to standard SAR protocol. Recovery ratios are indicated for the reference grain (on the left hand-side).
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simulation for a single, unique grain present on the disk, for comparison, which provided consistent results (recovery ratio of 1.03 ± 0.02 and test dose sensitivity change inferior to 5%). As expected, under the influence of another grain, the offset of the recovered doses of the reference grain (A) increases as the dose induced to the grain B increases and as the distance between the two grains reduces. When compared to the results obtained from the real measurements and for similar conditions (distance of 360 mm and doses of 20, 50 and 100 Gy given to grain B), we can note that these simulations yielded similar or higher offsets (1.17 ± 0.02, 1.20 ± 0.02 and 1.41 ± 0.03 versus 1.13 ± 0.02, 1.32 ± 0.02 and 1.46 ± 0.02, respectively). Thus, in all likelihood, cross talk from a nearby grain is the dominant factor here that hampers our ability to accurately measure a dose in grain A. 3.5. Evaluation of the cross talk effect During measurements based on a traditional SAR protocol, influence from surrounding grains will affect the determination of individual equivalent dose in two ways: in the natural measurement and for every laboratory-induced doses used to construct the growth curve. Based on our simulation results with two grains of identical brightness properties, we observe that the effect of an adjacent grain on the original growth curve is relatively limited, as the curve is only slightly displaced (Fig. 4). Thus, for a constant Lx/Tx ratio, it would yield to a maximum variation of the inferred equivalent dose of 0.82 Gy (Lx/Tx corresponding to an equivalent dose around ca. 20 Gy; 0.33 Gy of variation for Lx/Tx ca. 10 Gy) for various distance between the grains. The fact that the Lx/Tx varies little bears resemblance to the fact that laboratory-induced luminescence in quartz is relatively universal and standard in shape (Roberts and Duller, 2004). We record an increasing of the original equivalent dose of up to 7 Gy in our simulations (scenario with smallest distance between grains and maximum dose given to the influencing grain). Therefore, the main source of error is due to the
Fig. 5. Proportion by which the “natural” signal of the reference grain is increased, for different scenarios of initial dose given to the influencing grain and distance between them.
additional signal coming from the surrounding grain during the ‘natural signal’ measurement. In our simulation, this translates into an increasing of the original signal from 10% to more than 65% (Fig. 5), yielding to a variation of the initial Ln/Tn ratio from 1.87 to up to 2.70. However, these statements apply only for the very specific greatly controlled situation in this experiment (i.e. influence from only one grain, identical brightness properties, distance controlled). It is likely that the influence of cross talk on growth curve construction and especially initial signal measurements greatly increases for a more realistic situation when many grains are randomly spread on the aliquot with varying brightness properties and original doses (in the case of a partially bleached sediment). Finally, the simulations show that even keeping a much greater distance between grains (1080 mm) than the safety distance
Fig. 4. Influence of cross talk on the growth curve construction of the reference grain, for different distances with the influencing grain. The red curve represents the growth curve for a grain sitting all alone. The effect on the resulting equivalent dose is limited with a maximum variation of 0.82 Gy (upper right, Lx/Tx corresponding to ~20 Gy), and a variation of maximum 0.33 Gy in the area of the curve for a dose corresponding to around 10 Gy. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: Gribenski, N., et al., Investigation of cross talk in single grain luminescence measurements using an EMCCD camera, Radiation Measurements (2015), http://dx.doi.org/10.1016/j.radmeas.2015.01.017
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previously suggested (360 mm, section 3.2.) is insufficient to avoid a significant cross talk effect, in case of large difference of initial doses and brightness properties between the grains.
4. Potential solutions to counteract the effect of cross talk The experiments above have shown that performing single grain measurements with an EMCCD detector and using the AG2GUI advised method of image processing at its current stage of development does not allow obtaining reliable individual signal and equivalent dose estimates per grain. Additional procedures are therefore necessary to overcome the effect of cross talk influence from adjacent grains. Fig. 7. Recovery ratio (filled circles) of the reference grain (A) in relation to the dose given to the neighbouring grain (B). The results were improved with the use of the Background subtractor plugin (open circles).
4.1. Brightness criteria As a first possibility, one could try to apply an additional criteria based on the brightness of the grains, assuming that the brightest grains should be less sensitive to luminescence influence from surrounding grains. To test if such criteria could be relevant, we plotted the net ‘natural signals’ (averaged through ROI) of the single grains obtained from the first experiment (cf. section 3.1.) against their equivalent dose (Fig. 6a). The same was performed, but by using the net signals obtained for a 60 Gy regeneration dose (Fig. 6b). By this way, the signal can be approximated as a luminescence index since every grain received the same induced dose. In both cases, the majority of the data points are concentrated within a limited range of signal strength (from 25 to 100, for Fig. 6a and from 50 to 125, for Fig. 6b, analog-to-digital units) and the measured single grain doses are widely scattered, without any correlation between signal strength and the correct dose value recovered (10 or 50 Gy). For the data points exhibiting outstanding brightness, very few grains recover, indeed, the initial dose given. However, other grains with similar or slightly lower brightness properties are still widely spread between 10 Gy and 50 Gy. Therefore, these results show that discriminating grains based on their brightness properties to avoid single grain doses biased by cross talk is not efficient. In addition, applying such a procedure would lead to the exclusion of an important number of grains, thus reducing the appeal of an EMCCD approach. Hence, seeking corrective procedure to remove the effect of cross talk in every grain is preferable.
4.2. Potential of image processing algorithms: example of the “Background Subtractor” tool One potential solution is the use of image processing algorithms. Numerous algorithms, from simple to very complex, are widely used and constantly developed for processing images and analyzing light intensities of various objects in many different fields (such as micro-biology, medical sciences and photometry astronomy). For our purpose, we tested the potential of such approaches by using the Background Subtractor plugin software for ImageJ (Cardinale, 2010), which is based on a local histogram approach through a sliding square window (20 pixels here) to calculate and remove the local inconstant background illumination. By applying this algorithm on the luminescence images, we aim at reducing the blurring of the luminescent objects and so the cross talk effect, before integrating the signal and calculating the equivalent doses. First, we applied this additional step to the original image data sets obtained during the one-to-one grain real experiment (cf. section 3.3.). When compared to the initial recovered doses of the reference grain, we observe a clear improvement, with very satisfying recovery ratios (<10% offset) when the neighbouring grain received initial doses of 20 and 50 Gy (Fig. 7). The dose recovery test only still failed (recovery ratio of 1.22 ± 0.05) when a dose of 100 Gy is given to the neighbouring grain. The use of the Background Subtractor plugin to reduce the effects induced by cross talk seems relatively successful in that situation.
Fig. 6. The net natural signal (a) and the net signal of a regenerative dose of 60 Gy (b), averaged through the ROIs, are plotted against equivalent doses. Signal is given in analog to digital units (a.d.u.). No clear trend between brightness strength and recovered equivalent dose is observed.
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Fig. 8. Distribution of single grain equivalent doses obtained when applying an additional step of local background removal using the Background Substractor plugin on the luminescence images. (a) Distribution without any rejection criteria (n ¼ 124); (b) equivalent dose distribution when grains with recuperation ratios >0.1 or recycling ratios outside 20% of unity are rejected (n ¼ 47). In both cases, no peaks at 10 and 50 Gy are distinguishable.
However, it was necessary to test how it would perform in more realistic circumstances, when there is a multitude of grains of varying brightness separated by varying distances on the aliquot surface. For this, the plugin was applied to the image data set obtained during the first dose recovery test performed on aliquots with more than one hundred grains spread on the surface (cf. section 3.1.). The distribution of the single grain equivalent doses obtained is still widely dispersed, for both when all grains are considered (Fig. 8a), or when rejection criteria are applied (recuperation ratio >0.1, recycling ratio outside 0.8 and 1.2; Fig. 8b). No distinct grouping around 10 Gy and 50 Gy are observed, as it would be expected in the case of success. However, we note that the number of grains for which an equivalent dose could be calculated slightly increased (124 versus 104 grains, and 45 versus 27, respectively), and the uncertainties are reduced (relative error around 20e30 % in average instead of 40e70 %). The Background subtractor removes local background intensities by evaluating the frequency of pixel intensities for a given surrounding. It can be assumed that part of this background is given by the relatively flat cross talk signal. Hence, application of the plugin reduces the portion of additional signal coming from surrounding grains and reduces the uncertainty and the spreading of the normalized data points used for the growth curve reconstruction.
5. Discussion and conclusions The experiments carried out in this study show that grains spread over a flat surface without further precaution are clearly influenced by signal emitted by surrounding grains, when performing single grain EMCCD luminescent measurements. However, it is very difficult to quantitatively assess the effect of cross talk in general, as it will probably highly vary according to the samples being measured. Indeed, the influence of this phenomenon will be strongly modulated by the gradient intensity between grains, inherent to their brightness properties and/or to their accumulated dose. Besides, the effect will be amplified as each grain will be influenced by a cumulated signal emitted by numerous surrounding grains. Experiments showed that keeping a ‘safety’ distance between grains is insufficient in minimizing cross talk effect and counteracts the desire to have a sizeable amount of grains over the disk in order to maximize the statistical output of one aliquot. Introducing
brightness criteria to reject single grain doses more susceptible to be significantly biased by the phenomenon also proved to be inadequate. With regard to these elements, we consider the successful measurements of single grains spread on a flat surface while using an EMCCD camera cannot be performed without introducing an additional action to directly counteract or correct for the cross talk influence from surrounding grains. For this, several potential solution exist. The most straightforward is to set a physical barrier between grains by using a special sample carrier such as used by the Risø system (Thomsen et al., 2014). Although it probably will not totally prevent cross talk, as the effect is not only physically in nature, due to a lateral spreading of light once it comes of the grain, but also due to optical effects, such as blurring in the lens. In addition, the use of a disk with a fixed geometry (e.g. drilled holes) would negate the possibility to randomly spread grains on a flat surface, allowing, for example, the use of different grains sizes. Further development in the optical setup might as well reduce blurring. However, this would certainly be at the cost of a loss in sensitivity and in detection window flexibility. Finally, we showed that the results obtained in the simple situation when using only two grains with moderate brightness difference can greatly be improved by applying a relatively basic imaging processing tool. Therefore, we strongly encourage further exploration of image processing solutions based on more sophisticated algorithms. In case of success, this approach would open much more possibilities to use EMCCD cameras in luminescence dating, beyond the traditional measurement of well separated grains.
Acknowledgements We are grateful for very constructive reviewer's comments that helped us to improve the presentation of this study. We thank Krister Jansson for his valuable suggestions on how to improve the flow of the text, as well as for his continuous support and that of Arjen Stroeven and Jonathan Harbor. We also acknowledge Irina Rogozhina and Lucile Verrot for their patient advising in computer programming. This work was supported by a Swedish research Council grant to Arjen P. Stroeven and Krister N. Jansson (No. 20114892 and 2009-4411, respectively), and Sebastien Huot received funding from the CFI-FEI (Canada Foundation for Innovation) during this work.
Please cite this article in press as: Gribenski, N., et al., Investigation of cross talk in single grain luminescence measurements using an EMCCD camera, Radiation Measurements (2015), http://dx.doi.org/10.1016/j.radmeas.2015.01.017
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Please cite this article in press as: Gribenski, N., et al., Investigation of cross talk in single grain luminescence measurements using an EMCCD camera, Radiation Measurements (2015), http://dx.doi.org/10.1016/j.radmeas.2015.01.017