@@johanna.m.dela-cruz Dear Johanna, thank you for the response. After you set the threshold, you did not click the apply. So, after the clicking reset, could we measure the fluorescent intensity directly? Thank you.
@@merveemen3838 If you don't click Apply, the image is thresholded but not converted to a binary image. Measurements can be done directly on the previously thresholded image when the ROIs on the Manager are shown.
If I intend to do quantitative fluorescence analyses, should I refrain from adjusting contrast when taking the images? The software that controls the CCD allows the contrast adjustment (gramma value?); if I do that, will the fluorescence intensity of the sample relative to the background change? If so, when I use the default contrast level, are the measured intensities genuine values?
Hi Feline. Acquisition software usually have contrast adjustment for visualization purposes, but data values are unaffected. Post-acquisition processing like gamma adjustment is what you need to worry about when it comes to intensity measurements. Background subtraction should be fine as long as weak signals are not masked.
@@johanna.m.dela-cruz Thank you. It may depend on the acquisition software used. I used an Olympus DP7X camera and DP controller software. The contrast can be adjusted by changing the "input" value, which INDEED changes not only the absolute intensity values of objects but also the intensity ratio against the background. So now my question is if I left the "input" value as default (1), are the acquired intensities "genuine" and suitable for quantitative analyses? Thanks again.
@Feline Tropical Excitation light intensity and exposure time are ultimately what affect fluorescence signal. I do not know what the default settings are in your software, but you just need to make sure that signal is neither too weak nor too saturated. Increasing exposure time or light intensity will most definitely increase fluorescence intensity, and vice versa. The question about whether this is genuine or not depends on sample and application. Quantitative analysis (and comparisons) can be done on several images if they are acquired with the same settings.
Dear Johanna, thank you so much for the video. Does the calibration (pixel to micrometer) is required to measure fluorescent intensity? Thank you.
Hi @merveemen3838. Spatial calibration is not necessary for measuring mean gray value, but may be needed for integrated density measurements.
@@johanna.m.dela-cruz Dear Johanna, thank you for the response. After you set the threshold, you did not click the apply. So, after the clicking reset, could we measure the fluorescent intensity directly? Thank you.
@@merveemen3838 If you don't click Apply, the image is thresholded but not converted to a binary image. Measurements can be done directly on the previously thresholded image when the ROIs on the Manager are shown.
If I intend to do quantitative fluorescence analyses, should I refrain from adjusting contrast when taking the images? The software that controls the CCD allows the contrast adjustment (gramma value?); if I do that, will the fluorescence intensity of the sample relative to the background change?
If so, when I use the default contrast level, are the measured intensities genuine values?
Hi Feline. Acquisition software usually have contrast adjustment for visualization purposes, but data values are unaffected. Post-acquisition processing like gamma adjustment is what you need to worry about when it comes to intensity measurements. Background subtraction should be fine as long as weak signals are not masked.
@@johanna.m.dela-cruz Thank you. It may depend on the acquisition software used. I used an Olympus DP7X camera and DP controller software. The contrast can be adjusted by changing the "input" value, which INDEED changes not only the absolute intensity values of objects but also the intensity ratio against the background.
So now my question is if I left the "input" value as default (1), are the acquired intensities "genuine" and suitable for quantitative analyses? Thanks again.
@Feline Tropical Excitation light intensity and exposure time are ultimately what affect fluorescence signal. I do not know what the default settings are in your software, but you just need to make sure that signal is neither too weak nor too saturated. Increasing exposure time or light intensity will most definitely increase fluorescence intensity, and vice versa. The question about whether this is genuine or not depends on sample and application. Quantitative analysis (and comparisons) can be done on several images if they are acquired with the same settings.
nice format
Thanks, I appreciate it.
nice
Hi @Navnath. I hope it was helpful. Thanks for watching.