Tube Formation Analysis in the Automated Cellular Analysis System

Transcription

1 Tube Formation Analysis in the Automated Cellular Analysis System 1 ibidi GmbH, Version 1.0,

2 Table of Content Specifications... 3 Step-by-Step Guide... 4 Analysis... 6 Example Report Job ibidi GmbH, Version 1.0,

3 Specifications Input Compatible image formats Compatible video formats Image Size jpg, png, bmp, tif mp4, avi, mov 500 px x 500 px (min) 3840 px x 2160 px (max) Image aspect ratio Acceptable range is 4:3 to 16:9 Contrast As show in sample images below User Input Pixel size Time lapse frame rate Field of view User must input the pixel size in px/µm User must input the time between frames 0.5 mm to 2 mm Output Average perimeter µm Average area µm² Tube length µm Tube count number Total branching points number Data table Tab delimited format 3 ibidi GmbH, Version 1.0,

4 Step-by-Step Guide 1. Upload video/images to ACAS (either quick analysis or standard). Checking Multi-image upload enables selection of more than one picture by using the ctrl or shift key. On Windows systems, all pictures in a directory can be selected with the key combination ctrl + A. a. The system will automatically create and run an analysis job after the image files are uploaded. b. Once the analysis jobs are completed for all wells, a report job will be automatically created. 2. You will find a report in the experiment page (and also in the experiment-set page) typically 5 minutes after the final well has been uploaded. Each well will also contain a tracked movie (or tif file). ibidi GmbH, Version 1.0,

5 The ACAS system generates many types of output images. Output image collections of less than 31 frames will be stored in a multi-frame tif file. 31 or more images will be stored in an mp4 file. See ACAS User Guide for detailed information. 5 ibidi GmbH, Version 1.0,

6 Analysis TUBES, CLOSED LOOPS, AND BRANCHES An algorithmic attempt is made to find tubes, closed loops, and branch points. Image quality will significantly affect the accuracy of the feature identification. We recommend phase-contrast microscopy. We recommend a minimum resolution of 0.6 pixels/µm. ASSAY OPTION Assay options can be set by including the option in the experiment-set description field. ibidi GmbH, Version 1.0,

7 1. Colorizing Loops in Angiogenesis Tube Formation ACAS by default does not colorize the loops. You can enable this feature by adding the following code to the experiment-set description: COLORIZE-TRACKING-IMAGE Here is a sample of colorized closed loops: 2. Faint Edge Detection in Angiogenesis Tube Formation Analysis As the experiment progresses, the tubes become thinner until the point when the tubes visually disappear. During this transition the human reader can find faint tubes that are beyond the capability of automated analysis. In the image shown below, the red boxes indicate areas where the tubes are in various stages of disappearing from the image. The automated system must also face a large variety of input images of varying resolution and quality. An aggressive linear edge detection can introduce un-wanted artifacts in noisy images. So we have chosen to be less aggressive on faint edge detection, by default, to provide the best possible result for the maximum number of researchers. However, we have provided an option to increase the aggressiveness of the faint edge detection. 7 ibidi GmbH, Version 1.0,

8 In this example, a manual analysis indicates the presence of 21 closed loops. In this example, the default analysis mode (less aggressive faint linear edge detection) will produce the following output, finding a total of 15 closed loops: ibidi GmbH, Version 1.0,

9 To increase the aggressiveness of the faint linear edge detection, add this option to the experiment-set description field: FAINT-EDGE-DETECTION With this option enabled, the following output is produced, finding a total of 22 closed loops. While the total loops in this analysis are one more than the manual analysis, there are artifacts which missed one loop and created false loops. 9 ibidi GmbH, Version 1.0,

10 3. Excluding Loops which Flow out of the Image in Angiogenesis Tube Formation Some researchers prefer to include loops which are partially detected, others prefer to exclude loops which are only partially in the captured image. The ACAS default setting is to exclude partial loops. This can be overridden be setting this option in the experiment-set description: DONT-EXCLUDE-EDGE-LOOPS In this example, with manual analysis, we found 9 closed loops that are fully enclosed in the captured image. In automated analysis, seven closed loops are detected that are completely enclosed in the image, less the margin. A key distinction between the auto and manual analysis is the image margin. The algorithms are not able to accurately detect structures to the very edge of the image. The greater the resolution (increased number of pixels), the smaller the margin will be (that is, the algorithms will do a better job up to the edge. In this low resolution image (0.65 pix/µm), the margin is relatively large. ibidi GmbH, Version 1.0,

11 Example Report Job The tube formation report job is contained in an HTML file, with the option to download the tracking data in a.csv format. The report job screen is subdivided in four sections that can be viewed by a click on the section respectively. Information: This section contains overall information of the experiment, such as the name of the project, the title of the experiment set and experiment, as well as the experiment ID number. Settings: This section shows the settings that were defined in the experiment sets in order to summarize the setup and conditions for documentation purposes. 11 ibidi GmbH, Version 1.0,

12 Overview: This section contains the main results of the report shown in five different diagrams. The charts in this section are overlays of all positions (wells) of the experiment. The results are also summarized in a data table. ibidi GmbH, Version 1.0,

13 The corresponding data can be obtained by a click on the download icon ( is formatted in a.csv for use in your favorite analysis software. ). The data Please note that the charts look different depended whether your data is time lapsed. The report will display time data if it is available. Well Details: The last section lists the data of every position (well) separately. Choose a Well under if you want to study individual detailed reports for the corresponding well. Among other things the analyzed well images across time are shown. The view will change to metrics, curves and graphs for the selected Well only. The data that is previewed at the end of this section can be obtained as well by a click on the download icon ( ) 13 ibidi GmbH, Version 1.0,