Daniel:Notebook/RNAFISH/2014-9-16
Trial Run (Started 08/29/2014)[edit]
Microscopy[edit]
Threshold Comparison[edit]
- 20140916 RNAFISH FOXP2-546 CUX2-647.lif Production1 BestThreshold ch00.jpg
Field 1, Best threshold
- 20140916 RNAFISH FOXP2-546 CUX2-647.lif Production1 NoThreshold ch00.jpg
Field 1, no thresholding
- 20140916 RNAFISH FOXP2-546 CUX2-647.lif Production1 NoZeroThreshold ch00.jpg
Field 1, no zero threshold
This gallery brings up an interesting comparison for thresholding. What role does the threshold play? The three images are of the same field, same image, just with different thresholds. The first is the "best threshold", which uses what I determine to be an appropriate level of zero thresholding with the max threshold set such that all remaining pixels are very bright. The second is the image alone, with no thresholding performed at all. The third brings the max threshold down so that the pixels are bright, but has no zero threshold, meaning even pixels with value 10 and under show up. The "no zero" image shows that there is a considerable amount of overlap between the two channels, as almost all large spots of FOXP2 are also seen with CUX2, but at a much lower intensity. Why?
Sectional[edit]
The other image is a large sectional image, and brings up other questions.
One thing to note is that the stitching isn't great with the Leica software, since this was supposed to be a straight image. Of important note, however, is that there is no signal whatsoever near the top of this sectional. One thing I have noticed on these long sections is that the z-dimension can change quite a bit over the length of the sample. This may be the cause of the reduced signal, which is otherwise ubiquitous. We have not yet viewed any real patterns/layers in the fluorescence, which is what we're looking for, but I don't think this is layering, I think it is a microscope error.