Experiments on the operation of the micro image analyzer (Optimas 6.0)
Experiments on the operation of the micro image analyzer (Optimas 6.0)
Understand and master the principles and processes of image capture and analysis in immunohistochemistry experiments. (Neurobiology Laboratory Handout; edited by Du Jizeng and Chen Xuequn; 2004.09)
Operation method
Optimas 6.5
Principle
In immunohistochemistry experiments based on the principle of specific binding of antigen and antibody, due to the difference in the amount of antigen under the influence of different experimental conditions, the stained pictures of tissue slices of the same area from different animals will show differences in color depth. In image analysis, such differences in color depth are expressed as differences in gray scale integral values per unit area. The ultimate goal of image analysis is to semi-quantitatively compare the differences in tissue staining between control and experimental groups, and between experimental groups under different conditions, based on the differences in gray scale values caused by the depth of staining.
Materials and Instruments
Optical microscope; PixeLINK digital camera; computer (operating system windows; software optimas) Move 1. Place the stained piece under a light microscope and capture the image at the appropriate magnification using image capture software. Caveat Several key tools of Optimas in the analysis of immunohistochemical resultsWhen optimas is used to analyze immunohistochemistry images, there are several key software tools that must be highlighted.1. Target Area Selection Tools: After clicking on these tools with the mouse and dragging them over the image, you can select the area of interest in different shapes, such as square, indeterminate, or oval. 2. domain value adjustment tool: in the grayscale mode image color values, 0 means black, 255 means white. When we adjust the domain value, the computer according to the range of values we selected, select the image area in line with the selected area is called the foreground, showing yellow, unchecked, is the background.To adjust the domain value, the visible reference display standard is the domain value line. Tap the domain value adjustment panel, you can drag the upper and lower two scroll bars, the mouse to move, or manually enter the gray value to determine their own need to calculate the color domain value of the region. 3. Automatic area search tool: set the domain value, click on this tool, the software will be based on the domain value in the target area automatically generated to meet the requirements of the small area, circled in red. This area is also the area from which we will collect data. 4. Data Collection Settings: In this panel, you can set the type of data to be collected and the target software after data output. 5. Macro Recording Tool: With this tool, you can record the operations you need to perform repeatedly, and then run the macro every time you perform the same operation in the future. For more product details, please visit Aladdin Scientific website.
2. Open the image in optimas.
3. Convert the image to grayscale mode.
4. Select your target area for coloring, e.g. pvn or hippocampus or son.
5. Determine the field value.
6. directs the computer to select the eligible area based on the domain value.
7. transfers the area of the region, the optical density integral value, and other data to excel.
8. calculating the average optical density value of the target area you need in excel, and using the statistical functions it contains to derive the statistical values and draw statistical charts.