Fluorescence Microscopy (High-Content Imaging) SPP for the analysis of lipid droplets for the Discovery-1 automated microscope of Molecular Devices

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= 1. Input  =
= 1. Input  =
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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. Left side: green channel with lipid droplets Right side: red channel with nuclei and cytoplasm.<br>
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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. Left side: green channel with lipid droplets Right side: red channel with nuclei and cytoplasm.<br>  
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[[Image:Bodipy_image_green.jpg|300px]]
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Image of the green channel with stained lipid droplets  
Image of the green channel with stained lipid droplets  
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= 2. Analysis  =
= 2. Analysis  =
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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 To get also cell based data a newer version of this Software is currently tested.<br>
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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 To get also cell based data a newer version of this Software is currently tested.<br>  
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== 2.2. Journal Description  ==
== 2.2. Journal Description  ==
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<br>2. An intensity based treshold for light objects is applied to identify all the stained lipid droplets in the image  
<br>2. An intensity based treshold for light objects is applied to identify all the stained lipid droplets in the image  
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Image after the application of the sharpen image filter and the intensity based threshold.  
Image after the application of the sharpen image filter and the intensity based threshold.  
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<br>3. Regions are created around the object for analysis of lipid droplets  
<br>3. Regions are created around the object for analysis of lipid droplets  
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<br>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.  
<br>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.  
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<br>Data analysis with the integrated morphometry analysis in MetaMorph
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<br>Data analysis with the integrated morphometry analysis in MetaMorph  
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== <br>2.3. Output parameters<br> ==
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== <br>2.3. Output parameters<br> ==
Well based parameters:  
Well based parameters:  
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Area of each lipid droplet and average area per image<br>Average intensity of pixels within regions
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Area of each lipid droplet and average area per image<br>Average intensity of pixels within regions  
Intensity Standard Deviation  
Intensity Standard Deviation  
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Intensity Signal/Noise ratio  
Intensity Signal/Noise ratio  
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Minimum and Maximum Intensity
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Minimum and Maximum Intensity  
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Bild Excel  
Bild Excel  
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= 3. Data processing  =
= 3. Data processing  =

Revision as of 10:59, 24 April 2009

Contents

1. 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. Left side: green channel with lipid droplets Right side: red channel with nuclei and cytoplasm.

 



Image of the green channel with stained lipid droplets

 

2. Analysis

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.


2.2. Journal Description

1. 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


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









Bild










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



3. Regions are created around the object for analysis of lipid droplets




Bild


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.



Bild



Data analysis with the integrated morphometry analysis in MetaMorph


2.3. 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

 

 

 

Bild Excel

 

3. 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


4. Proposals and discussion points

Write here any proposals and discussion points you may have.

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