Very nice explanations! Thank you Johanna. One thing I would still add to the issue of weighted vs. unweighted conversion, which per se destroys realistic measurements is that the initially chosen pseudo-color (LUT) additionally influences the intensity measurement. If for example a single channel is saved with a specific pseudo-color applied as RGB image the mean intensity changes dependent on the color choice. Moreover, channels can only be splitted properly if the image consists of maximally 3 channels which contain pure red, green or blue colors. If one or more fluorescent channels are displayed in other colors such as cyan, magenta, yellow, ... a proper separation is not possible anymore (similar to the image indicated at minute 8:10 for a non-fluorescent image)
![Measuring Protein Expression and Cellular Fluorescence [Mean Gray Value vs Integrated Density]](http://i.ytimg.com/vi/GaRxdBkoqlQ/mqdefault.jpg)
![Measuring Protein Expression and Cellular Fluorescence [Mean Gray Value vs Integrated Density]](/img/tr.png)
![FIJI (ImageJ): Bone Analysis with BoneJ2 [Trabecular Bone]](/img/n.gif)
Very nice explanations! Thank you Johanna.
One thing I would still add to the issue of weighted vs. unweighted conversion, which per se destroys realistic measurements is that the initially chosen pseudo-color (LUT) additionally influences the intensity measurement. If for example a single channel is saved with a specific pseudo-color applied as RGB image the mean intensity changes dependent on the color choice. Moreover, channels can only be splitted properly if the image consists of maximally 3 channels which contain pure red, green or blue colors. If one or more fluorescent channels are displayed in other colors such as cyan, magenta, yellow, ... a proper separation is not possible anymore (similar to the image indicated at minute 8:10 for a non-fluorescent image)
Thanks for your input, @janbrocher6824. I appreciate it.