Confocal Laser Fluorescence Microscopy to Measure Oil Concentration in Produced Water: Analyzing Accuracy as a Function of Optical Settings

ments); more specifically, testing for compliance with discharge regulations by measuring the oil in post-treatment produced water. The microscope can potentially be used to view, count, and analyze the oil droplets in treated produced water to estimate the concentration of oil in a particular sample. Such calculations can be done by image processing techniques to interpret the stacks. Calibration of the CLFM method involves comparison of estimated oil content with the CLFM to a prepared sample with known oil content. This normalized comparison refers to the percent recovery (CLFM estimated content/known content) and the standard deviation of the percent recovery to assess accuracy and precision, respectively. Several settings on the CLFM affect the intensity of the fluorescence in the images produced, and thus, affect the concentration of oil that is calculated. One study utilized the CLFM for geochemical analysis of cave deposits and addressed this issue of fluorescence intensity by maintaining all settings constant in an effort to normalize the fluorescence intensity measurements (Orland et al., 2014). None of the previous studies with CLFM, however, have delineated a clear relationship between a sample oil concentration, the number of optical sections per stack, the quantity and location of stacks, the percent recovery, and the standard deviation. This is largely due to the lack of a systematic method in retrieving confocal image data. The objective of this research is to establish a strategy for representative sampling and identify patterns between the sample concentration, number of optical sections per stack, quantity and location of stacks, threshold value for grayscale to binary image processing, percent recovery, and standard deviation. This reINTRODUCTION The confocal laser fluorescence microscope (CLFM) enables viewing fluorescing objects in samples and creating 3D images by optical sectioning. The study by Wilson (2011) showed that the function of the CLFM is similar to that of a conventional widefield optical microscope, but the confocal uses spatial filtering techniques to reduce information from the background, rendering higher quality images. The study demonstrated that the CLFM has the capability to eliminate secondary fluorescence from areas outside of its set focal plane by allowing light to pass only through a pinhole. The 3D images are produced in stacks that are a compilation of optical sections which are lateral images of the cross-sectional area of the specimen at each particular point on the z-axis. The predominant application of the CLFM since its introduction has been in life sciences. However, recent novel studies are investigating the feasibility of CLFM for subsea applications (subsea engineering refers to oil and gas extraction from oceanic environConfocal Laser Fluorescence Microscopy to Measure Oil Concentration in Produced Water: Analyzing Accuracy as a Function of Optical Settings