Puncta Analysis: Measurement of Puncta Per Cell

Firstly, slices of each 15 μm z-stack were overlaid into a single image for puncta analysis using Fiji software (extension of ImageJ version 1.53c) (Schindelin et al., 2012). For each image, the unit of measurement was converted from pixels to microns using the scale bar, followed by converting the image to an 8-bit format. The MorphoLibJ plugin (Legland et al., 2016) was then used, with the morphological filter ‘White Top Hat’ to reduce background fluorescence that can obscure puncta size (Qvarnström et al., 2004). Next, the images underwent thresholding to isolate the puncta visible in the original image (Figure 2). Thresholds were kept consistent across experimental groups within each replicate. Finally, the number of puncta was assessed, producing a numbered list of each puncta and its associated area in μm². For each oocyte, the output for the number of puncta was sorted by size in Microsoft Excel, and the total size distribution was compared between groups in GraphPad Prism 8.4.3 (San Diego, CA, United States). Size categories assessed were defined by the relevant vesicle labeled by each marker along with additional size categories for other puncta detected to assess all potential differences within each dataset. These size categories included the recorded autophagosome size (Mizushima et al., 2002) (0.5–1.5 μm in diameter or 0.196–1.767 μm² in area) for LC3B and recorded lysosome size (Ponsford et al., 2020; Trivedi et al., 2020) (0.03–0.5 μm² in area) for LAMP1. Additional puncta detected outside of these categories were assessed in arbitrary categories increasing by 1 μm² up to >10 μm² for LC3B and BECN1, and increasing by 0.1 μm² up to >1 μm² for LAMP1. The differences between experimental groups for each category were then determined through statistical analysis as detailed below.

Details of the puncta analysis method utilized to analyze puncta size distribution within immunolabeled oocyte images. (A) Firstly, the single-channel image of antibody staining to be analyzed with a scale bar of known size was acquired. (B) The scale bar was traced using the straight-line tool, and the scale of the image was adjusted from pixels to microns (Analyze > Set Scale > Input scale bar size as known distance). The scale bar was then circled and deleted using an area selection tool to remove from the image. (C) The image was converted to an 8-bit grayscale format for further analysis (Image > Type > 8-bit). (D) White top hat transformation (Plugins > Morpholibj > Morphological filters) was then performed. To do this, the ‘White top hat’ operation was selected, and parameters were adjusted to obtain a reduced background and enhance features of interest. (E) The image was then thresholded (Image > Adjust > Threshold) by selecting the threshold that most accurately represented the detail of the initial image (T = 20 here). (F) Finally, the ‘analyze particles’ feature was selected (Analyze > Analyze particles) to produce a dialog box containing the number and size of each puncta measured. This information was copied into Excel, where puncta were sorted by size and categorized as desired.