Sometimes this doesn't happen or you want to change the overall contrast to look at dimmer objects. This is important for being able to scroll through the stack and see the spectral trends. By default, ImageJ should set the contrast as constant for all channels if you have >6 channels in your image. Typically a spectral image is imported into ImageJ as a hyperstack with sliders for channel, z, and time if present. I provide these tools for circumstances under which accessibility to microscope manufacturer's software is not available (in all other circumstances the manufacturer's tools are superior). įirstly, let me note that ImageJ is not the ideal tool for interactive spectral exploration. The second data set is a cropped version of Figure 5S published here. Throughout this tutorial I will show examples with data available here: fluocells© and here: pombe. There are going to be a lot of tools here, so I am not going to list menu paths for them but rather suggest you use the Fiji command finder to find the plugins. Our typical workflow proceeds from interactive data exploration to spectral measurement and finally to quantitative linear unmixing. Here I will describe a number of tools we have created at Stowers for exploration and analysis of spectral imaging data in Fiji/ImageJ. Spectral imaging is a powerful tool for a wide variety of applications including autofluorescence exploration, isolation and verification of signals weaker than autofluorescence, demultiplexing of many dye labels, and Forster resonance energy transfer (FRET).
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