TIRF imaging of lysosomal exocytosis

 1\ Training Dataset (120 movies)

This dataset contains the data used for the training and the evaluation of ExoDeepFinder (i.e. the 120 movies presented in figure 1D). Each folder corresponds to a single TIRFM movie of one RPE1 VAMP7-pHluorin transfected cell. All cells are in control conditions i.e. no drug treatment. Importantly, some movies are from paired experiments, before-after treatment, but only conditions before treatment have been included in the dataset. Hence, the mentioning of a drug in the folder name is a reference to the control before the aforementioned treatment and does not indicate drug treatment. While most of the cells are seeded on fibronectin-coated coverslips (62 movies), some of them are seeded on different substrates:

-Cells from folders with the reference “Micropattern Ring” were seeded on fibronectin coated, ring-shaped micropatterns (diameter of 37µm and thickness of the adhesive ring of 7µm) (17 movies).

-Cells from folders with the reference “Rectangles pattern” or “Micropattern rectangle” were seeded on fibronectin coated, rectangle-shaped micropatterns (9x40µm) (18 movies).

-Cells from folders with the reference “PLL coating” were seeded on PLL coated coverslips (23 movies).

Moreover, while most of the cells are only VAMP7-pHluorin transfected (96 movies), some of them have been co-transfected:

-Cells from folders with the reference “Paxillin” were co-transfected with paxillin-mCh (19 movies).

-Cells from folders with the reference “RFP-Rab6A” were co-transfected with mCh-Rab6A (5 movies).

Each folder contains a sequence of .TIF images corresponding to each frame of the movie. Movies can be opened with ImageJ as an image sequence. Each movie is made of 1001 16-bit images. The pixel size is 0.160µm. In addition, each folder contains a .nd file that can be opened with ImageJ. Moreover, a .tif cell mask is associated to each movie (“Flood fill” to fill holes inside the mask and “Clear outside” algorithms are not applied on these masks but necessary to obtain simply connected masks).

Lastly, each folder contains a .txt file with the manual annotations of exocytosis events, considered as the ground truth reference. This .txt file is an output of imageJ. The x,y and t coordinates of exocytosis events are given, respectively, by the columns “X”, “Y” and “Slice”. This file reports x and y coordinates in pixels and time as frame number.

Finally, the dataset contains an excel file (.xlsx) giving for each file:

-Folder name

-The total duration of the movie (based on the actual computer saving times of the frames and not the theoretical time set by the microscope)

-The number of frames

-The invert of the frame rate

-The background i.e. average signal in a region outside the cell at t=1

-The signal i.e. average signal in a region inside the cell at t=1

-The classical SBR, computed as the ratio of the two previous columns

-The exocytosis SBR, computed as the ratio between peak intensity of the event (F) and background before the exocytosis event (F₀) (see paper methods)

-The SBR level, i.e. the classification as low, medium or high SBR based on the exocytosis SBR rank

-Group (either “training” or “inference”) to specify if the movie has been used for ExoDeepFinder training or evaluation

2\ Robustness Dataset (90 movies)

This dataset contains the data used for the robustness evaluation (figure 2). It contains 4 sub-datasets. Each sub-dataset is made of several folders containing .TIF images corresponding to each frame of the movie, the corresponding .nd file, a .tif cell mask and a .txt file with the manual annotations of exocytosis events, considered as the ground truth reference. This organization is the same as found in the “Training Dataset”. In addition, each sub-dataset contains an excel file (.xlsx), giving for each folder i) the name, ii) number of frames, iii) the invert of the frame and iv) SBR, similarly to the excel file in the “Training Dataset”.

2.1\ Bafilomycin Dataset (32 movies)

This dataset corresponds to RPE1 cells transfected with VAMP7-pHluorin, seeded on fibronectin-coated coverslips and treated with Bafilomycin A1 (at 100nM for 1h). Each cell is imaged before and 1h after the treatment. The following table shows the pairing.

2.2\ Histamine Dataset (34 movies)

This dataset corresponds to RPE1 cells transfected with VAMP7-pHluorin, seeded on fibronectin-coated coverslips and treated with histamine (at 100µM). Each cell is imaged before and immediately after the treatment. The following table shows the pairing.

2.3\ HeLa Dataset (14 movies)

This dataset corresponds to HeLa cells transfected with VAMP7-pHluorin and seeded on fibronectin-coated coverslips.

2.4\ CD63 Dataset (10 movies)

This dataset corresponds to RPE1 cells transfected with CD63-pHluorin and seeded on fibronectin-coated coverslips.