3. It is necessary to analyze and analyze immune fluorescence images. This step requires a certain understanding of the principle of immunology, such as different types of antigen or antibodies can produce different fluorescence signals, which can be compared by these signals to ensure that there is no specific antigen or antibody in the sample.
Body.
4. Interpret and report based on analysis results so that doctors or other researchers can understand the situation in the sample. If further research is needed, immune fluorescence images can also be combined with other experimental data such as PCR, western blot, etc. To facilitate further research.
imagej How to measure immune fluorescence images?
Explore the quantitative fluorescence magic of ImageJ: Average Fluorescence Intensity Detection Details
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In biological research, immune fluorescence coloring is the golden tool for revealing the distribution of cell-specific proteins. However, with this powerful image analysis software, ImageJ, we can not only perform morphological observations but also fine semi-quantitative analysis of protein expression, the key is to measure the average fluorescence intensity.
defines a single-channel fluorescence image, the gray value of each pixel is exactly the reflection of its fluorescence intensity. Calculate the average fluorescence intensity of a particular region, the formula is as follows:
After understanding this basic concept, ImageJ provides us with fine operating steps to this process.
instance demonstration
Let us practice fluorescence images under a laser co-focus microscope. First, open the image and select a single channel (Image-Color-Sp
lit Channels), if RGB format, remember to separate channels. For high-precision 16-bit or 32-bit images, threshold can be adjusted directly, but keep as much signal information as possible to avoid loss.
Then use Image-Adjust-Threshold to set thresholds and select default values to reduce human error. Make sure that only the signal area is selected and avoid the proportion scale (may affect the outcome). Select the most suitable threshold algorithm, such as Default, but if necessary, try other methods to optimize the selection
In Analyze-Set Measurements, the key is to select the mean gray value and limit to threshold, which will ensure that the measurement is the average within the threshold. Click the measurement (Analyze-Measure), the result will show the mean, which is the desired average fluorescence intensity.
Attention and practical tips
Although the average fluorescence intensity is quantitative analysis, but in the immune fluorescence experiment
Various factors (such as exposure, focus gap) can lead to errors. Therefore, standardizing the operating process and setting fixed measurement conditions are critical. In addition, unlike the average fluorescence intensity and optical density, ImageJ processing does not require Invert or Calibration, usually presented in Arbitrary Units (AU) as units.
If you have problems using ImageJ, you can contact me at any time, mailbox: zhaoyc9@163.com . For more practical tutorials, please follow my ImageJ
A practical tutorial column that will help you improve the efficiency of experimental analysis.
Let us master this powerful tool together to enhance the accuracy of quantitative analysis of immune fluorescence and provide strong data support for scientific research. Wish you a breakthrough in the experiment!
ImageJ practical techniques--Average fluorescence intensity detection (quantitative analysis)
In an immune fluorescence coloring experiment, half-quantitative analysis of specific protein expression can be done by detecting the average fluorescence intensity in addition to morphological analysis.
The detailed steps of ageJ for the detection of average fluorescence intensity are as follows:
I, defined with the principle
Average fluorescence intensity: For a single-channel (single-color) fluorescence image, the gray value of each pixel represents the fluorescence intensity of the point.The fluorescence intensity of a specific region can be calculated by the formula: Average fluorescence intensity (Mean) = the total fluorescence intensity of the region (IntDen) / the area area (Area).II, operating step
If the image is in RGB format, the target channel must be separated first. In ImageJ, select “Image-Color-Split Channels”.
If the image is already in 8bit, 16bit or 32-bit format, the next step can be done directly. Note that 32-bit and 16-bit images can contain more pixel range, encoding more information, avoiding conversion to
8bit to reduce information loss.
adjust the threshold value
select "Image-Adjust-Threshold" to adjust the threshold value. The software will automatically select a default value, in order to eliminate the errors caused by the hand-selecting threshold of different photos, it is best to use the default threshold.
the essence of setting the threshold is to select as many signal areas as possible, without selecting the background. Red range is the selected range, proportional scale and other irrelevant areas need to be adjusted by Threshold or after selecting the field Edit-F
Remove.
Please note that the “Dark Background” is selected because the background of the fluorescent image is usually black.
If the threshold set by the default algorithm does not meet the requirements, you can select “Image-Adjust-Auto Threshold” to try other algorithms.
Select the appropriate threshold algorithm
If the threshold set by the default algorithm does not meet the requirements, you can select “Image-Adjust-Auto Threshold” to try other algorithms.
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1 Select “Try all”
After clicking OK, all algorithms set thresholds will be listed and the appropriate algorithm will be selected according to the results.
Set the measurement parameters
Select "Analyze-Set Measurements" to set the parameters that need to be measured.
Confirm "Mean gray value" and "Limit to threshold" (very important), click OK.
The measurement is the average fluorescence intensity of the entire image.
Detects the average fluorescence intensity
Select "Analyze-Measure" for the detection.
Click on Measure and the detection results pop up, where "Mean" is the average fluorescence intensity (Mean is the mean gray value, equal to IntDen/Area), IntDen is the total fluorescence intensity.
can be copied to Excel or directly exported to CSV
Fourth, instance display (the following image shows the key interfaces and results in the above operating steps) Through the above steps, we can use ImageJ to detect the average fluorescence intensity of the immune fluorescence image, thereby providing strong support for semi-quantitative analysis of specific protein expression.
rary Units (AU).