First steps

The following tutorial will provide an overview of the fundamental concepts and functions of ImageC, offering guidance on the initial steps required for effective utilization of the software. The tutorial will demonstrate the following:

• ImageC Welcome window

• Creating a new project

• Adding channels

• Starting an analyzes

• Open analyzes results

• Export results

Analyze images

Note

If you have problems running ImageC see here.

Running ImageC

Once ImageC has been successfully launched, the user will be directed to the start wizard. At this point three options are available: create a new project, open an existing project, open the results of a previous run.

ImageC start screen

By clicking the New project button a project wizard is opened. Enter basic information on your project and group your images.

ImageC new project wizard

Title	Description	…
Scientist name	Name of the person who is responsible for this analysis.	Optional
Organization	Organization responsible for the analysis.	Optional
Working directory	Storage Directory of the ‘to be analyzed’ images.	Mandatory
Order of Images in well	If images are taken from in a (6, 12, 24, 96, 384) well format, the order of the images position in the well can be determined here.	“Optional
Group by	Images may be left ungrouped, or can be grouped by Filename regex or Directory.	Mandatory
Filename regex	If Images are grouped by filename, the regex should indicate the order of the images: Regex to extract plate row, plate column and image index from the	image filename.
Regex test	Used to test the regex settings. Enter your Image Name and see if the wells are recognized. in the regex test result
Regex test result	Result of the regex applied on the text given in the regex test field.
Notes	Some free text notes on the experiment.

Caution

Make sure that the grouping options and regex settings are correct, as they are needed for valid image sorting and mean well infos.

Regex

Filename grouping uses regex (regular expressions) to extract the position on the plate and the position of the image in the well from the image filename. Extracted are: plate row and column position and the image index in the well.

It es expected that the row position is a character in the range of [A-Z] and the column and index a decimal number. A typical series of file names for the regex _((.)([0-9]+))_([0-9]+) might look something like the following:

name_A1_1.tif name_A1_2.tif name_A1_3.tif name_A1_4.tif name_A2_1.tif name_A2_2.tif name_A2_3.tif name_A2_4.tif

To experiment with regular expressions, have a look at regex101.

It is important to set the correct grouping options in combination with the correct filename regular expression. Based on these settings, ImageC performs calculations of the results during a running analysis. This dramatically improves the speed of report generation. However, if the grouping settings are wrong, these statistics will also be calculated in a wrong way.

When grouping by Foldername or Filename is selected ImageC will pre-calculate the statistics based on the determined group. Average, Median, Min, Max, Standard deviation, Sum and Count are calculated for each group When opening a analysis result (see dive into the tutorials) these pre-calculated values are loaded for a fluid and fast view.

A change of the grouping settings after analysis is currently not supported by ImageC. If the grouping settings are changed the analysis has to be repeated.

The Working Directory should be set to the folder where the images to be analyzed are stored. ImageC will perform a recursive folder search with the selected Working Directory as the base folder to find all supported image files. All found files are listed in the Overview panel.

Overview Panel

Once ImageC has been successfully launched and the new project wizard has been closed via the Apply button, the Overview panel is displayed.

ImageC overview panel

The overview panel displays the options for analysis of a channel in the middle of the screen. With the Add image channel buttons, new channels can be added.

On the right hand side the parsed OME meta data from the selected image is shown.

A list of all images found in the selected working directory is displayed in the bottom toolbar. If a BIG TIF image is used, the image is subsequently cut into smaller tiles and the single tiles are displayed next to the image.

Adding a channel

By clicking Add Image Channel a new channel is added to the analysis settings. Up to 10 channels can be added. All predefined EVAnalyzer pipelines are included in this version. Select EV channel for loading a pipeline (preprocessing, object filtering, detection) optimized for EV quantification from single vesicle imaging images with low background. Select Cell brightfield for loading a pipeline (preprocessing, object filtering, detection) optimized for cell detection on brightfields images. Select Nucleus for loading a pipeline (preprocessing, object filtering, detection) optimized for nucleus detection after fluorescent labelling of the nuclei (e.g. Hoechst, DAPI). Select EV in cell for loading a pipeline (preprocessing, object filtering, detection) optimized for EV quantification in complex material like cells. Select

Setting with one added channel

When the analysis is started, each defined channels of all images found in the working directory are processed.Non defined channels are not processed. The results for each channel are stored in a file based database with the extension .duckdb for later reporting generation and statistics.

By clicking on a channel, the channel editor is opened. The channel editor allows to specify channel meta data, preprocessing steps, detection settings as well as filters for objects and images.

Channel editor

Defined template settings are stored as JSON template files and stored in the ./templates directory beside the ImageC binary.

At the right hand side a live preview is displayed. It shows the resulting object detection after all applied preprocessing steps and filters. Changing a parameter will directly change the preview, enabling a fast and easy adjustment and fine-tuning of the settings. A live object count is displayed at the bottom of the image. Based on image size and the complexity of the selected preprocessing algorithms it could take a couple of seconds for refreshing the preview. The preview can additionally be zoomed in and out and a second window with the original image and the processed image side by side further enables smooth detection setting.

Hint

See the section Channels for detailed information about the channel settings parameter.

Starting the analysis

With the back button on the top left the overview panel is displayed again. After all channels are added and all needed channel settings are done, the analysis can be started by pressing the button on the top.

Analysis running

A dialogue box informs you about the progress of the analysis. At the bottom right of the dialog a Open results folder button is placed. Press this button to open the file explorer showing the folder with results of the actual analysis run.

With Stop button a running analysis can be interrupted. It may take a couple of minutes to stop a running analysis since all still in progress tasks have to be finished.

Press the Close button to close the dialog after a successful finished analysis run.