From LipidomicsWiki

Contents 1  Input 2 Analysis with the MetaMorph Software (Version 6.1) 2.1 Journal Description 2.2 Output parameters 3 Analysis with the granularity application module of MetaXpress 2.0 3.1 Output parameters 4 Data processing 5 Proposals and discussion points

 Input

Automated analysis of lipid droplets requires two microscopy channels to get well and cell based data. In the first, green channel, the lipid droplets stained with Bodipy 493/503 are detected and in the second, red channel cell nuclei and cytoplasm stained with HCS CellMask Red Cytoplasmic stain are detected. From each well of a multi-well plate, the microscope takes 4 fields per channel. These 384 images are saved as tif files. As an example, the two corresponding images of one field are shown below.

 

Green channel with stained lipid droplets	Red channel with nuclei and cytoplasm

 

Analysis with the MetaMorph Software (Version 6.1)

To analyse the primary images with the MetaMorph Software (Version 6.1) a Journal was written to get well-based primary parameters. To get also cell based data a newer version of this Software is currently tested.

Journal Description

• An sharpen median filter (preprocessing filter) is used to improve the images for better identification, separation and definition of objects (lipid droplets) as distinct elements

• An intensity based treshold for light objects is applied to identify all the stained lipid droplets in the image

Image after the application of the sharpen image
filter and the intensity based threshold.

• Regions are created around the object for analysis of lipid droplets

Image with regions around lipid droplets

With the integrated morphometry analysis in MetaMorph the output parameters of the analysis are defined and logged into an excel file. Also different graphical presentations of the data can be made with the integrated morphometry analysis.

Data analysis with the integrated morphometry analysis in MetaMorph

Output parameters

Well based parameters:

• Area of each lipid droplet and average area per image
• Average intensity of pixels within regions
• Intensity Standard Deviation
• Intensity Signal/Noise ratio
• Minimum and Maximum Intensity

Example of an Excel Table containing well based parameters

 

Example of a summary table of well based parameters

 

Analysis with the granularity application module of MetaXpress 2.0

Backgrounds of images analyzed with the granularity application module are processed using the Adaptive Background CorrectionTM system. This system automatically corrects uneven image background throughout the image by adapting to local content. This allows for more robust segmentation and analysis repeatability and enables to specify the detection sensitivity as intensity above local background. A gray level value is selected that represents the least bright granule minus the background intensity near the granule. The software estimates the background for each lipid droplet locally to correct for cases of images with uneven background. The same procedure was applied for the nuclei and the value of the background is subtracted from the value of the nucleus. To differentiate nuclei from non-nuclear material the appropriate minimum and maximum lipid droplet width in microns (µm) is selected. Lipid droplets less than the specified minimum width will be considered as noise patterns. In addition also the appropriate minimum and maximum nuclei width were selected to select the nuclei for segmentation.

 

 Lipid droplet and nuclei segmentation for cell based data

Cell-based data are generated	and well-based data are generated

 

Output parameters

This application module detects, counts and measures granularity and nuclei characteristics and provide well and cell based data.
The following parameters are detected:

Lipid droplets per cell (average) – represents the numer of all lipid droplets in one well related to the number of nuclei in this well

Mean lipid droplet area - demonstrates the mean lipid droplet area of all lipid droplets in one well related to the number of nuclei in this well

Average lipid droplet intensity - adds together the average intensity of pixels within the stained area in all cells of one well (4 fields per well) and averages them

For graphing the mean values of these parameters from each well were further calculated into mean values of those wells with the same treatment. 

Data processing

These primary parameters are further processed in Microsoft Excel to show the average intensity, the average area and the total count of lipid droplets

Proposals and discussion points

Write here any proposals and discussion points you may have.