Since the initialization algorithm in CNMF fails due to the large contaminations from the background fluctuations in this setting (recall Figure 2), we use the ground truth as its initialization. We found that it was more effective to initialize each spatial footprint by regressing the initial neuron traces onto the raw movie data (see Materials and methods for details). These pixels are defined as seed pixels. For the first experiment, with the exception of red spruce (Picea rubens Sarg.) Also, P⁢() and L⁢() have to be updated after each neuron is initialized, but since only a small area near the initialized neuron is affected, we can update these quantities locally to reduce the computational cost. (1997) indirectly calculated the quantity of CaOx crystals in developing soybean seeds by quantifying total and soluble oxalate and assuming that the difference between the two fractions was insoluble oxalate present in the form of CaOx. obtained via GRIN lenses; we hope to present these results in future work but decided against including these results here to avoid lengthening an already-long paper. Extraction Pure calcium metal can be made by the same method used by Davy. The paper is very clearly written, and the simulations backed up by the in-vivo imaging data clearly demonstrates that this technique is by far superior to any other currently being used, including ICA/PCA or other variations. The small amount of water-soluble oxalate possibly included sodium hydrogen oxalate, potassium hydrogen oxalate and some free oxalate (Hönow and Hesse 2002). PCA/ICA took ∼211 seconds to converge, using 250 PCs and 220 ICs. A 0.6 mm diameter GRIN lens was implanted above the injection site within the BNST. However, total Ca may not be the most relevant measure to evaluate Ca sufficiency for physiological functions because a part of the total Ca is irreversibly committed to structural components of cells or tied up as CaOx and is not pertinent to a plant’s present metabolic needs. For each extracted neuron A⁢(:,k), we first convert it to a 2D image and perform morphological opening to remove isolated pixels resulting from noise (Haralick et al., 1987). In this case, most neurons inferred by PCA/ICA were inferred by CNMF-E as well, with the exception of a few components that seemed to be false positives (judging by their spatial shapes and temporal traces and visual inspection of the raw data movie; detailed data not shown). This is because the pure NMF model in CNMF does not model the true background well and the residuals in the background are mistakenly captured by neural components. This analysis is consistent with our observations that background dominates the fluorescence signal and extracting high-quality neural signals requires careful background signal removal. We have edited the text significantly to clarify a number of details that were not sufficiently clear in the original manuscript. The samples were quantified using a Series 200 Perkin-Elmer HPLC system using 20 µl injections, and UV/VIS detector (Perkin-Elmer Corporation, Norwalk, CT, USA); absorbance was measured at 215 nm. After choosing the thresholds for seed pixels (Figure 3H), we run CNMF-E in full automatic mode, without any manual interventions. Since PCA/ICA seeks statistically independent components, which forces the temporal traces to have near-zero correlation, the correlation structure is badly corrupted in the raw PCA/ICA outputs (Figure 4D). Bottom left: the residual of the raw video (top left) after subtracting the estimated background using CNMF-E. Top middle and top right: the spatiotemporal activity and temporal traces of three neurons extracted using CNMF-E. Bottom middle and bottom right: the spatiotemporal activity and temporal traces of three neurons extracted using PCA/ICA. The red lines are the thresholding boundaries for screening seed pixels in our initialization step. We now turn to the analysis of large-scale microendoscopic datasets recorded from freely behaving mice. 10, Extraction of Calcium in the Presence of Iron (II) after;v ' MhsM Cyanide , , ® 38 ' viii. The authors should instead just say "non-convex" without the word "jointly". This preprocessing removes most calcium transients by replacing those frames with the previously estimated background only. A Python implementation has also been incorporated into the CaImAn toolbox (Giovannucci et al., 2017b). While the reviewer agrees with the authors that "s_i is completely determined by c_i and G^{(i)}, and B^f is not optimized explicitly", nonetheless s_i and B^f are optimization variables. We start by assuming that neurons with high spatial overlap do not fire spikes within the same frame. However, for extractions with acetic acid, there was more variation for Ca compared with oxalate among the 10 species. This method is very computationally intensive and some direction needs to be given on how to run the software to allow a large number of movies to be analyzed in a reasonable amount of time. Both Ca and oxalate quantities extracted in water varied within a relatively small range for all species (see Figure S2 available as Supplementary Data at Tree Physiology Online). However, H2SO4 resulted in higher calcium extraction efficiency as compared to HCl and HNO3. (D) Fluorescence traces (raw data; true and estimated background; true and initial estimate of neural signal) from the center pixel as selected in (B). 8) It is mentioned in subsection “in vivomicroendoscopic imaging and data analysis” that manual interventions were applied in the data analysis shown in this paper. Thus, (P-All) is simply incorrect as written. Nociceptin neurons in the bed nucleus of the stria terminalis regulate anxiety, Action planning and action observation in rodent parietal cortex, Encoding the relationship between anxiety-related behaviors and nociceptin neurons of the bed nucleus of the stria terminalis, Data from: efficient and accurate extraction of in vivo calcium signals from microendoscopic video data. As for the first iteration of the model fitting, we set each Bit−=1|Ωi|∑j∈ΩiX~jt as the mean of the X~j⁢t for all j∈Ωi. Relationship between the amount of calcium and oxalic acid extracted sequentially in: water (a), then acetic acid (b) and finally in PCA (c) or HCl (d) from pooled foliage from a single tree from each of five conifer and five hardwood species. Fresh foliage tissue (300 mg) was placed in 10 ml of ddH2O and subjected to three freeze–thaw cycles. Approximately 0.2 g of each tissue sample was digested in 9 ml of concentrated nitric acid in Teflon vessels using a microwave (MARS Xpress, CEM Corporation, Matthews, NC, USA) following EPA method #3052 (Microwave assisted acid digestion of siliceous and organically based metrics, 1996). In some cases, X might include large residuals from the inaccurate estimation of the neurons’ spatiotemporal activity A⁢C, for example, missing neurons in the estimation. 2004). Exploring and understanding the mechanisms that control the formation of CaOx and release of Ca from these crystals in plants is vital to discern the relationship between Ca bioavailability and forest health (White and Broadley 2003). Bottom left: the estimated background. The field of view was 253×316 and the number of frames was 2000. (C) The spatial and the temporal cosine similarities between the ground truth and the neurons detected using different methods. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. As for the initialization of a single neuron given a seed pixel, it is only (O⁢(T)). (2009) reported a qualitative method for the extraction and determination of CaOx crystals from croton (Codiaeum variegatum) that required large quantities of leaf tissue. Bottom middle: the denoised neural signals. Figure 2C shows that the fluorescence traces of the center pixel and its neighbors are highly correlated due to the shared large background fluctuations. The variability observed among trees within a species or different species with regard to the concentration of Ca, oxalate and CaOx crystals may be due to the phenology of growth, site chemistry and innate differences among species. In step 1, the time cost is mainly determined by spatial filtering, resulting in O⁢(d⁢T) time. Bottom: the filtered fluorescence trace at the seed pixel or the initialized temporal activity (both raw and denoised). There were 53 components initialized. Details are included in the revised manuscript (subsection “Ranking seed pixels”). See the new panel B in Figure 4, and also our responses to Essential revision question (Carvalho Poyraz et al., 2016). Calcium is first selectively extracted by organic acid leaching, followed by carbonation of the leachate to precipitate CaCO 3.

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