Using just four SP-D molecules, with arms plotted as hexamers, and recorded as trimers (reversing the mirror symmetry and using the N term value as the whole N term peak width), the glycosylation peak (now called the 3rd set of peak(s) in the trimer) sometimes has a lumpy appearance, and signal processing programs routinely divide that single (sort of) largest of peaks (second to the N term and the CRD peaks) into separate adjacent but clearly contiguous peaks, sometimes as many as 6.  When i see the brightness areas i dont often agree on what the signal processing programs call “separate” peaks as they really come together in one single significant elevation, clumped as separate but connected peaks.  The number of separate peaks within what is typically pointed out as the glycosylation peak in AFM micrographs actually has more than one peak… these data sort out using 4 dodecamers (16 trimer plots – assessed by 5 signal processing programs, several imaging processing filters, and one unprocessed image collectively) that about 2 peaks show up in the glycosylation “bright” spot.   1.92+0.204.  This will likely change with the addition of more molecules added to the set.

Below are the numbers for peak width for the glycosylation peak. The characteristics of peak, that is whether it is a single simple peak or a peak with multiple elements,  I dont find good mention of in the literature.

It is worth noting that because there presumably are three sites (one per strand of the trimer) which are potentially glycosylated, and whether there could be a bumpiness to the bright areas in an image representing the glycosylation peak. This might reflect structural features of “image” the three single strands if they are or are not glycosylated while wound in to a trimer thus causing a displacement of glycosylation sites perhaps showing up as peaks within the glycosylation peak (envision three beads on three separate cords, wound tightly, each displaced slightly creating a lumpy look.  This is completely different than the images which show the large CRD and neck elements which do show large bright areas quite distinctly separated at either end of the hexamer.  see CRD peak appearance with AFM images, left, and possible completely glycosylated trimer on right (NOT TO SIZE).

In most micrographs there is a dominant “bright” region which has been identified as the glycosylation peak in numerous publications, including the one by Arryoy et al which i rely upon heavily) as a single peak. Close visual examination reveals a couple of things, 1) it is a peak of varying width, and 2) sometimes it is possible to see small sub peaks within the whole. Signal processing and peak counts from images before and/or with the application of image filters, it is common to see bright spots within what might be considered a single larger bright region (brightness in this context is higher on the gray scale value y axis than lower). Defining the “modest but colvincing ” number for peaks along a trimer has been the focus of this research. Both total relative peak heights and valley to valley widths (not taking into account slope or mid peak areas) have yet to be determined for a hexamer of SP-D.

So using just a half dozen analysis protocols on FOUR (4) SP-D dodecamers (16 trimers – where the entire N term peak is included in each trimer), the first three peaks (beginning at the whole N termini junction peak) looks like this. N=19.86nm+1.46, tiny peak=2.53+0.74, glycosylation peak width=8.4+0.69.

My tendency was/is to see the AFM images of this molecule as having 15 peaks. Two pominent CRD peaks at either end of the hexamer, one peak (possibly with its own central valley (so moving the odd number of peaks to an even 16) in the middle and two recognized peaks either side of the N term. These peaks are numbered in many of these blog posts as Peak 1=N term, two tiny peaks on either side of 1, mirrored as peaks=2, then the two glycosylation peaks =3 (either side and lateral to the tiny peaks and sometimes clustered peaks.  Peaks which follow I am now suggesting are “real events along the collagen like domain”  as peak 4,  moderate size, fairly wide peaks, next very narrow low peaks=5, broad low peaks=6, and sometimes visible peak which appears to be the coiled coiled neck domain where sometimes it is covered by the CRD, sometimes not=7, at the end, the CRD peaks which are very prominent sometimes exhitibing clustered peaks=8. These I have colored with specific colors through out the peak counting process, as my own assessment of which of these categories the signal processing peak programs miss the mark (which sometimes they do, sometimes they dont).

Not all trimers show all peaks, thus the attempt to find a mean width, peak height and humber amont the dozens of AFM images I have collected.

Here is a link to description of the ridge plot (Joy plot) shown below.

The length of a trimer was determined  from the center of the N termini junction peak to the edge of the carbohydrate domain (since this is a lumpy molecule…. the most central route for the line (plot) through the CRD was used. Many examples of the type of line are given in previous posts.

Applications were AS BEFORE- No processing,  gaussian blur (5 or 10px) and gaussian blur with limitrange (100 (or above)-255), and each is then subjected to all of the following signal processing applications for peak detection: Lag 5, Threshold 1,  Influence 0.05 (batch process), Scipy (Prominence 2, Distance 30, Width 5, Threshold -, Height -), Octave, autofindpeaksplotx,y and iPeaksM80, and excel template PVDTxlsx smooth 11.  This means that there are two hexamers (four trimers) each with no processing, some processing, and each of the latter subjected to 5 different signal processing routines.  Some select other processing can be included.

Trimer widths in nm, as all lumped values, individual values, and as a single group of four.

The number of peaks per trimer is calculated with all independent measures, as molecule measures (that would be each trimer of four individual SP-D images). The N term peak is calculated in full, WITH each N term peak that is not divided into two peaks. So adding up the peak number — keep in mind that N is counted twice if it is a single large peak.

Previously, using dozens of image and signal processing programs for literally hundreds of plots, of a single SP-D image (41_aka_45), the number of peaks per trimer was 8. Check out the post here. It is very encouraging to have a selected set of image and signal processing programs provide almost identical results to that original single molecule dataset. That means to me that the gaussian blur and limit range imaging filters can be used somewhat confidently to provide easier counting of peaks along an arm of a hexamer (or trimer) of SP-D.

Just analyzing the left hand side of each of the four images, the trimer length in nm is different (as relates most likely to preparation artifact, in stretching or folding of the arms.

just the righ hand side of the image (the second trimer in the hexamers to be traced are as follows.

There are so many ways to sum these arms up i just decided to do them separately as 16 trimers comprising 4 dodecamers.  It makes little difference that i can detect and a nice conservative number of 145 nm as the usual hexamer length, and  73 for the usual trimer length (not exact yes… ). but counting more molecules might be more efficacious than deliberating on just four.

Two conclusions. 1)  Image processing is helpful, signal processing to count peaks, not so much.  The plots smoothed with the gaussian blur and enhanced with the limit range function are easy enough to manually count peaks, and the peaks counted manually are guided by ones knowledge which the current algorighms are not (as in not recognizing symmetry, and not permitting small peaks to follow big ones. etc).  When a few more molecules are counted and added to the list then perhaps I will find someone who knows how to “train” peak counting algorighms.  LOL.

2) The number of peaks per hexamer is likely to be between 13 and 15. THis is much greater than that proposed by Arroyo et al, who found 5.

Four dodecamers of SP-D, plotted in ImageJ, with and without gaussian blur, limit range image processing, and processed again with signal processing algorithms to determine peaks per hexamer.
Applications were – No processing or gaussian blur (5 or 10px) and gaussian blur with limitrange (100 (or above)-255), and each is then subjected to all of the following signal processing applications for peak detection: Lag 5, Threshold 1,  Influence 0.05 (batch process), Scipy (Prominence 2, Distance 30, Width 5, Threshold -, Height -), Octave, autofindpeaksplotx,y and iPeaksM80, and excel template PVDTxlsx smooth 11.
Four dodecamers were included in this set of numbers below…. analyzed as a total input, and as an N of four. Not a big difference.  This number is not too far off the extensive search for peaks in just ONE dodecamer (which was chosen for its microscopic appearance), where the peak number per hexamer was about 15,

Hexamer width, using image and signal processing, whether examining all hexamer plots independently or as summaries.  Very close numbers for nm length.