Nd 6.5 mg/mL MHE treated 5dpf zebrafish embryos. Notice the increased amplitude in the MHE treated fish which corresponds to an increased stroke volume (SV) B. and C. SV, heart rate (HR), cardiac output (CO) and ejection fraction (EF) measurements for Fourier method (B) and segmentation method (C). SV and EF were significantly increased according to both measurement paradigms (* indicates P,0.05, n = 22, 26 for Control and MHE treated fish respectively). doi:10.1371/journal.pone.0052409.gIn our initial hypercholesterolemia screen we were able to detect a decrease in mean fluorescence intensity in ezetimibe treated fish compared to controls. However, this difference is not as substantial as the difference between control and ezetimibe treatment in our manual screen [18]. 23977191 The reason for this discrepancy is inhibitor likely that in the automated Opera screen, data from the entire fish is acquired, including from the gut. As fish appear to eat a similar amount of food between treated groups and controls, this creates similar fluorescence output in the digestive tract in these groups. Therefore, it is likely that our assessment of mean fluorescence intensity over the entirety of collected images in each well le to this discrepancy between the magnitude of ezetimibe’s effect between manual and automated screen. The effect of fluorophore in the gut was minimized by allowing a 16 hour interval between feeding and imaging where fish had no access to food. This was to ensure that a maximal amount of fluorophore was expelled from the gut before imaging. Also, the area of the well imaged was optimized to capture as little of the intestine as possible. The influence of fluorescent cholesterol in the gut nevertheless has potential to decrease the sensitivity of the screen. However, this occurrence 
is also beneficialin its potential to identify both compounds that decrease intravascular cholesterol levels by inhibiting cholesterol absorption and compounds that facilitate the expulsion ofcholesterol. Hawthorn extract had a dramatic effect on BODCH fluorescent output compared to Epigenetics controls and in a dosedependant fashion (figure 2B and 2C). This agrees with our longer-term studies to determine the effect of whole ground hawthorn leaves and flowers on intravascular cholesterol levels [18]. Previous attempts to automate the analysis of cardiodynamic data in zebrafish employed the analysis of brightfield images of the heart beat [24] and have derived measurements of heart beat rhythmicity 
from Fourier power spectrum representations of blood flow in the caudal vasculature [25]. Compared to brighfield imaging, high-speed confocal data has the advantage of providing high contrast between the organ and surrounding tissue, greatly simplifying the automated detection of heart movements. Analyzing the heart beat for cardiodynamic data with the method presented here, opposed to extracting cardiac parameters from measurements in the vasculature [25], allows the extraction of not only frequency measurements, but also measurements of stroke volume and ejection fraction which indicate the inotropic state of the heart. However, the rhythmicity analysis presented in [25] provides a powerful tool for detecting the arrhythmic effects of drugs.Automated In Vivo Hypercholesterolemia ScreenIn order to validate our two automated analysis methods, we tested the accuracy of both techniques in determining stroke volume compared to manually measured waveforms (quantified by measuring the peak.Nd 6.5 mg/mL MHE treated 5dpf zebrafish embryos. Notice the increased amplitude in the MHE treated fish which corresponds to an increased stroke volume (SV) B. and C. SV, heart rate (HR), cardiac output (CO) and ejection fraction (EF) measurements for Fourier method (B) and segmentation method (C). SV and EF were significantly increased according to both measurement paradigms (* indicates P,0.05, n = 22, 26 for Control and MHE treated fish respectively). doi:10.1371/journal.pone.0052409.gIn our initial hypercholesterolemia screen we were able to detect a decrease in mean fluorescence intensity in ezetimibe treated fish compared to controls. However, this difference is not as substantial as the difference between control and ezetimibe treatment in our manual screen [18]. 23977191 The reason for this discrepancy is likely that in the automated Opera screen, data from the entire fish is acquired, including from the gut. As fish appear to eat a similar amount of food between treated groups and controls, this creates similar fluorescence output in the digestive tract in these groups. Therefore, it is likely that our assessment of mean fluorescence intensity over the entirety of collected images in each well le to this discrepancy between the magnitude of ezetimibe’s effect between manual and automated screen. The effect of fluorophore in the gut was minimized by allowing a 16 hour interval between feeding and imaging where fish had no access to food. This was to ensure that a maximal amount of fluorophore was expelled from the gut before imaging. Also, the area of the well imaged was optimized to capture as little of the intestine as possible. The influence of fluorescent cholesterol in the gut nevertheless has potential to decrease the sensitivity of the screen. However, this occurrence is also beneficialin its potential to identify both compounds that decrease intravascular cholesterol levels by inhibiting cholesterol absorption and compounds that facilitate the expulsion ofcholesterol. Hawthorn extract had a dramatic effect on BODCH fluorescent output compared to controls and in a dosedependant fashion (figure 2B and 2C). This agrees with our longer-term studies to determine the effect of whole ground hawthorn leaves and flowers on intravascular cholesterol levels [18]. Previous attempts to automate the analysis of cardiodynamic data in zebrafish employed the analysis of brightfield images of the heart beat [24] and have derived measurements of heart beat rhythmicity from Fourier power spectrum representations of blood flow in the caudal vasculature [25]. Compared to brighfield imaging, high-speed confocal data has the advantage of providing high contrast between the organ and surrounding tissue, greatly simplifying the automated detection of heart movements. Analyzing the heart beat for cardiodynamic data with the method presented here, opposed to extracting cardiac parameters from measurements in the vasculature [25], allows the extraction of not only frequency measurements, but also measurements of stroke volume and ejection fraction which indicate the inotropic state of the heart. However, the rhythmicity analysis presented in [25] provides a powerful tool for detecting the arrhythmic effects of drugs.Automated In Vivo Hypercholesterolemia ScreenIn order to validate our two automated analysis methods, we tested the accuracy of both techniques in determining stroke volume compared to manually measured waveforms (quantified by measuring the peak.
