Category Archives: Methods to assess TEM and AFM images

“peak” or not to “peak”

This is an example of why i get frustrated with peak detection apps…. vertical axis is grayscake 0-255, x axis not numbered is nm (which I have to calculate against a bar marker on the original micrograph) and is not really relevant here.

Peaks that are found are clearly marked at the point of valleys on each side, by the program (it is an excel program called PeakValleyDetectionTemplate.xls) and what this smooth function (smooth 11) thinks is a valley is highlighted in pink…. so i didn’t miss anything…. or add anything.

My draw program has a “snap too” function that snaps to the valley lines detected so I don’t even make the judgements in identifying those vertical valley indicator lines there so this program measures width between peaks with the lines (which I colored pink) and are easy to follow. BUT…

My ADDED arrow on the left points to a peak that I certainly would have called a peak that was not detected by this app, my added arrow on right points to a peak that I certainly would NOT have called a peak which this app thought was a peak (I could not even find a drop in grayscale of one single pixel).

Just out of wanting to know the facts in this study of SP-D, and trying to help determine the structure of the common multimer of SP-D (the dodecamer), I dont change those chosen valleys from the apps, I just report the results whether I agree with them or not. But sometimes these apps “think” (lol) too hard about lag, threshold, smooth, incidence, width, deviation, and influence etc…. and gloss over the obvious, not able to consider chance, inherent variability, possible radial and bilateral symmetry etc……..therefore at this point they are really not as good (in the method that I am using them) as human judgement (mainly mine ha ha ha) in my humble opinion. I also accept that this is a simple molecule, bilateral, with maybe 11-17 peaks routinely found. So its not a massive chaotic mess, but sometimes they just dont work.

Eight dodecamers: SP-D – CRD peak height and valley

These data are the pretty final. The next dodecamers will be added as individuals only, not as the sum of the long list of plots (e.g. 508 trimer plots) but using only an N of a dodecamer (which seems to me to produce the best data. None of these data have had the  width measurements standardized to a trimer length in nm (which will be done last).  I guess it is possible to standardize the grayscale values as well.

Eight dodecamers: SP-D – N term ?x subpeak height

There is often a slight decrease in the N term peak, likely where the N term domains join the trimers at the center of the dodecamer. This is an infrequent event and its detection is also related to how one draws the plot line through the center of the image of the molecule.  When the N term peak width is listed there are occasions when it interferes (reduces) the total N term peak width. The tiny change in grayscale (height and valley) of the N term peak is pretty small. I have put a ? in the title of this peak to denote its inconsistent appearance.

It is nevertheless listed here in a graphic and below, it appears in two of the eight trimers in these data.



Eight dodecamers: SP-D – carbohydrate recognition domain peak width

Same MO,  but since this peak appears 100% of the time (so far) there is no second column where the mean (and other parameters) is calculated excluding the missing (undetected) peak values.  Difference in CRD peak width is certainly not very different from the first 6 dodecamer summary than the first 8 dodecamer summary. Some benefit is gained by using the means of each, rather than the individual trimer values.