This is the user manual for the graphical interface for segmenting yeast images using the YeaZ convolutional neural network. You can find the training set with the segmented training images here: https://www.quantsysbio.com/data-and-software/.

Want to try out the neural network without installing any software? Check out our online segmentation tool at https://lpbs-nn.epfl.ch/.

The software requires a standard computer with enough RAM to apply the neural network. 8 GB RAM is enough to predict the 700 x 500 px image provided as the test data. The RAM requirements scales linearly with the number of image pixels.

It was tested on OS X High Sierra (10.13.6), Windows 10 Education, and Ubuntu 18.04.4.

Package dependencies: The convolutional neural network relies on Keras with TensorFlow. The Hungarian algorithm is implemented in the munkres package. In addition, standard image processing and scientific computing libraries are used.

Installation time is less than 5 minutes.

1. Clone this repository ("git clone https://github.com/lpbsscientist/YeaZ-GUI").
2. Download the parameters for the neural network:
3. Download the parameters for segmenting phase contrast images from: https://drive.google.com/file/d/1UTivmx_aEMpeGdOkCZO1CS9mcdJ3zmw2. Put the file in the folder /unet.
4. Download the parameters for segmenting bright-field images from: https://drive.google.com/file/d/1oxZpbJOwXxH6g453lCrBkdPB_2KD6rNo. Put the file in the folder /unet.
5. If you don't have conda or miniconda installed, download it from https://docs.conda.io/en/latest/miniconda.html.
6. In the command line, create a virtual environment with python 3.6.8 with the command conda create -n YeaZ python=3.6.8.
7. Activate that environment using conda activate YeaZ.
8. Install the necessary packages using pip install -r requirements.txt.
9. Run the program from your command line with python GUI_main.py

Using the neural network to make predictions is very memory intensive. This can lead the computer to run out of memory, which in turn causes the program to abort. However, the amount of memory that is needed depends on the size of the image. So, you may try cropping your images into several smaller images or removing empty space around cells if you do not have enough memory. For instance, using a 2015 MacBook Pro with 8GB of RAM and a 2.9GHz Intel Core i5 CPU, we were able to predict images of size 700 x 500 pixels.

If small buds aren't recognized as cells in your image, this is likely linked to a segmentation parameter that is too high. Relaunching the CNN with a smaller parameter will likely yield better results.

In case you only want to use the functionalities of the convolutional neural network and the segmentation, but not the full GUI, you only need the files unet/model.py, unet/neural_network.py (for making predictions), unet/segment.py (for doing watershed segmentation) and unet/hungarian.py (for tracking), as well as the weights for the neural network which have to be in the same folder. You can create predictions using the prediction function in neural_network.py (note that before making predictions, you have to use the function equalize_adapthist from skimage.exposure on the image). The segmentations can be obtained with the segment function in segment.py, and tracking between two frames is done using the correspondence function in hungarian.py.

Our CNN was trained on fewer cells with the bright-field technique (3841 unique cells imaged with 6 different exposure levels versus >8000 for phase contrast).

Jordan Xiao of Stanford University pointed out that one of the python modules (Pyqt5) has some issues on Mac OS Big Sur. Check out https://stackoverflow.com/questions/64833558/apps-not-popping-up-on-macos-big-sur-11-0-1 and https://stackoverflow.com/questions/64818879/is-there-any-solution-regarding-to-pyqt-library-doesnt-work-in-mac-os-big-sur/64856281 for more information and possible fixes.

Upon starting the program, it prompts you to select your images and your (new) mask file. There are two ways to select images: One can directly select a single image file using the button Open image file. Currently, we support Nikon .nd2 files and multi-stack .tif files in addition to all standard formats such as .png, .jpg, or .tif. One can also select a folder of image files using the button Open image folder. This takes every single image in the folder to be a frame in a timelapse recording of yeast cells. The folder must contain images that all have the same size.

The program will save segmentation masks in .h5 files. You can either create a new file by specifying its name in the text box or if you already have an h5 file for a specific set of images, you can select it using Open mask file. Note that when using an existing .h5 file, you also have to use the same images as you did at its creation.

After clicking OK in the launcher, the main program will open. It consists of an image display of three images, the current image in the middle, the previous one to the left and the next image to the right. This display was chosen to easily be able to check whether the cell numbers and the segmentations are consistent throughout time.

On the very bottom of the program, there is a status bar which displays the state of the program. In addition, when hovering over a button, it gives detailed information about what the button does and how to use it.

On the top left of the three images, there are multiple buttons that allow the user to use pan and zoom to navigate through the image. The home button to the very left allows to reset zoom and pan, while the arrows allow to go to the next and previous pan/zoom positions.

On the bottom of the images, there are three rows of buttons. The first row of buttons allows the user to select the field of view, color channel, and time frame. Note that support for multiple fields of view and color channels is currently only available when opening .nd2 files. The second row contains tools to make edits to the mask, in order to correct the mistakes of the CNN. Moreover, it allows to show or hide the segmentation mask and the cell numbers. On the bottom right, the buttons allow to launch the CNN, to recalculate the tracking, and to extract the fluorescence or segmentation masks.

After opening a new image file, the next step is typically to launch the convolutional neural network with the Launch CNN button. Note that if you are using a file with multiple color channels, this has to be done with the brightfield channel visible. This will open a dialog box, in which you can specify the time frames that you want to launch the CNN on as well as the field of view you want to use.

Moreover, it lets you specify two parameters: The threshold value specifies the predicted value above which a pixel is considered to belong to a cell. This value is set at 0.5 per default and doesn't have to be changed in our experience. Increasing this value will decrease the sizes of the cells and can come in handy if the cells tend to exceed their borders. The segmentation parameter tells the program how far away two cell centers have to be at least, in order for two cells to be considered as separate entities. It has to be adjusted depending on how large the image resolution is: For small resolutions, a value of 2 seems to work well, whereas 5 is good for higher resolutions.

After the CNN has run, it is possible to correct the mistakes it has made. This can be done in the following ways:

Add region: Add a region to an existing cell, by defining a polygon. First left-click on the cell to which you want to add a region, then continue clicking to draw a polygon to add. Finally click on the button again to confirm.

New cell: This works similar to Add region but defines a new cell. Draw a polygon using left-clicks and re-click the button to confirm.

Brush: This is an alternative way to add to a cell. The user right-clicks to select the cell to draw. Then the user can click and drag to draw the cell. The brush size can be controlled using the spin-box to the right.

Eraser: This can be used to remove a region from a cell. The use is the same as for Brush.

Exchange cell IDs: This allows correction of cell ID values by switching two cells.

Change cell ID: This allows changing the ID number of a cell. **Important: **If you change the number to the number of another cell, those two cells will from now on be considered as one single cell. This is useful for fusing cells that were oversegmented but has to be used with care.

Retrack: Having made edits to a frame, the cell numbers may no longer correspond in the next frame. This can be automatically fixed by navigating to the next frame and clicking the retrack button.

After the masks are satisfactory for a given field of view, you can click on the Extract button to export the results. There are two ways the results can be exported: Firstly, the masks that were found can be exported as multistack TIFF files. Secondly, the fluorescence together with several statistics giving information about every individual cell can be extracted and saved as a .csv file.

Moreover, you may only be interested in a subset of the cells visible in the image, which is why this second window allows you to select the cells which you are interested in. You can select or deselect multiple cells by drawing a polygon around them and confirming with a right-click, or select and deselect single cells by just left-clicking them. Note that the image that is displayed corresponds to the last frame for which you have a mask. Cells which disappeared throughout the timelapse video - and thus are not part of the mask - will be exported as well but indicated with a flag in the exported fluorescence csv.

When you want to extract fluorescence, you can add files from which to extract fluorescence by clicking the Add button. There you can either add a single image file, a multistack TIFF file, or a folder containing image files. Note that if multiple files or frames are used, the fluorescence file or folder must have the same amount of images as the image given to the program at startup.

The output csv contains one line for every combination of cells, timeframe, and channel. This allows the file easily to be read into pandas, and avoids a varying amount of columns depending on how many fluorescence channels are used. The following statistics are exported: The area of the cell, the mean intensity, the intensity variance, the total intensity, and the x and y coordinates of the center of mass. Moreover, the major and minor axes of the cells are found using principal component analysis. In particular, the angle of the major axis to the x axis is given, together with the length of the major and minor axis, thus, fully specifying an ellipsoid approximatiing the cell. Finally, we report whether the cell disappears, i.e., whether or not it is present in the last frame.

We guide you step-by-step through the demo:

1. In the startup dialog, click Open image file and select the file in the example_data folder from within the file dialog.
2. Give a name to the h5 mask file, such as example_data.h5. Press OK to confirm.
3. We want to predict the segmentation. Click Launch CNN. In the pop-up dialog. Enter 0 and 0 as the time bounds. Select the Field of View 1 by clicking on it. Press OK.
4. Wait for the neural network to predict. This takes about 1 min on a standard computer.
5. Now go through the image to verify the predictions and correct mistakes as needed. Instructions about how to use every tool is shown in the status bar at the bottom when you hover over the tool button.
6. Click Extract if you are satisfied. Extract the mask or the cell statistics, specify a file name, and you're done! In case you wanted additional fluorescence channels or only extract a subset of cells, you could also do this here using the corresponding buttons.

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