AMEBaS: Automatic Midline Extraction and Background Subtraction of Ratiometric Fluorescence Time-Lapses of Polarized Single Cells

Cell polarity is a fundamental biological process in which the concerted action of a collection of spatially concentrated molecules and structures culminates in the establishment of specialized morphological subcellular domains¹. Cell division, growth and migration rely on such polarity sites, while its loss has been associated with cancer in epithelial tissue-related disorders².

Apically growing cells are a dramatic example of polarity, where the polarity site at the tip typically reorients to extracellular cues³. These include developing neurites, fungal hyphae, root hairs, and pollen tubes, where multiple cellular processes show pronounced differences from the tip of the cell toward the shank. In pollen tubes, in particular, actin polymerization, vesicle trafficking, and ionic concentrations are markedly polarized, showing tip-focused gradients⁴. Pollen tubes are the male gametophytes of flowering plants and are responsible for delivering the sperm cells to the ovule by growing exclusively at the apex of the cell at one of the fastest growth rates known for a single cell. The tip-focused gradients of ions such as calcium⁵ (Ca²⁺) and protons⁶ (H⁺) play a major role in sustaining pollen tube growth, which is essential to accomplish its main biological function that culminates in a double fertilization^5,6. Thus, quantitative methods to analyze the spatiotemporal dynamics along the midline of apically growing cells are essential to investigate the cellular and molecular mechanisms underlying polarized growth^7,8,9. Researchers often use kymographs, i.e., a matrix that represents the pixel intensities of the cell's midline (e.g., columns) through time (e.g., rows), which allows visualizing cell growth and migration in the diagonal (Figure 1). Despite their usefulness, kymographs are frequently extracted by manually tracing the midline, being prone to biases and human errors while also being rather laborious. This calls for an automated method of midline extraction that is the first feature of the pipeline introduced herein named AMEBaS: Automatic Midline Extraction and Background Subtraction of ratiometric fluorescence time lapses of polarized single cells.

In terms of experimental procedures, quantitative imaging of ions/molecules/species of interest in single cells can be achieved with genetically encoded fluorescent probes¹⁰. Among the ever-expanding choices, ratiometric probes are one of the most accurate since they emit different fluorescence wavelengths when bound/unbound to the molecules of interest¹¹. This allows for correction of the spatial heterogeneity in the intracellular concentration of the probe by using the ratio of two channels with their channel specific background subtracted. However, estimating the background threshold for each channel and time point can be a complex task since it often varies in space due to effects like shading, where the corners of the image have luminosity variation relative to the center, and in time due to fading of the fluorophore (photobleaching)¹². Although there are multiple possible methods, this manuscript proposes determining the background intensity automatically using the segmentation threshold obtained with the Isodata algorithm¹³, which is then smoothed across frames through polynomial regression as a standard. Spatial components stemming from fluorescence heterogeneity unrelated to the target cell removed in¹², however, were ignored by this method. Automatic thresholding can be performed by several methods, but the Isodata algorithm produced the best results empirically. Thus, automatic background value subtraction and ratiometric calculation are the second main feature of AMEBaS (Figure 1), which, taken together, receives as input a stack of dual-channel fluorescence microscopy images, estimates the cell's midline and the channel-specific background, and outputs kymographs of both channels and their ratio (main output #1) after background subtraction, smoothing, and outlier removal, together with a stack of ratiometric images (main output #2).

AMEBaS was tested with fluorescence time lapses of growing Arabidopsis pollen tubes obtained under a microscope, either with Ca²⁺ (CaMeleon)⁸ or pH (pHluorin)⁶ ratiometric sensors expressed under the pollen-specific LAT52 promoter. Images from each channel were taken every 4 s coupled to an inverted microscope, a front-illuminated camera (2560 pixels × 2160 pixels, pixel size 6.45 μm), a fluorescence illuminator, and a water immersion objective lens 63x, 1.2NA. Filters settings used for CaMeleon were: excitation 426-450 nm (CFP) and 505-515 nm (YFP), emission 458-487 nm (CFP) and 520-550 nm (YFP), while for pHluorin, excitation 318-390 nm (DAPI) and 428-475 nm (FITC), emission 435-448 nm (DAPI) and 523-536 nm (FITC). A complete data set was added for testing at Zenodo (DOI: 10.5281/zenodo.7975350)¹⁴.

In addition, the pipeline was tested with root hair data, where imaging was performed with a light sheet microscope (SPIM) as previously described^15,16 with Arabidopsis root hairs expressing the genetically encoded Ca²⁺ reporter NES-YC3.6 under the control of the UBQ10 promoter¹⁷. The home-made LabView software that controlled the camera acquisition, sample translation and shutter of the light sheet microscope permitted the observation of the two cpVenus and CFP channels, but also the visualization of their ratio in real time. Every ratio image of the time-lapse represented a maximum intensity projection (MIP) between the cpVenus and CFP fluorescent channels images obtained from 15 slices of the sample spaced 3 μm apart. The time-lapse cpVenus/CFP ratio of MIPs was saved and directly used for the AMEBaS analysis.

Although this pipeline can work with multiple types of growing and migrating cells, it was specifically designed to analyze growing cells that grow exclusively at the tip, such as pollen tubes, root hairs, and fungal hyphae, where there is a correspondence of the non-growing cytoplasmic regions between frames. When such a correspondence is not present, the user should choose the complete_skeletonization option in step 1.3.1.1 (see the Discussion section for more details).

Figure 1: An overview of the pipeline workflow. The AMEBaS pipeline analyses and processes microscopic time lapses in three main steps: Single-Cell Segmentation, Midline Tracing, and Kymograph Generation. Please click here to view a larger version of this figure.