It appears that there are directional lines and background noise that can cause significant peaks to appear in LUT plots. THe ways that I think that one can counter these are:
1. determining whether there is isotropy/anisotropy Most clearly the lines in an example below show that measurements along .
2. Orientation (as well as image processing) impacts measurement since all these molecules (hundreds of them) have been observed in countless different positions, fallen by chance into as many different configurations influenced by preparation, pH, molecule density, interaction between different parts of different adjacent molecules (e,g, N termini with CRD, CRD with those from other molecules), multimer number, and positions with regard to — staining techniques, shadowing techniques, warping of the surfaces, and variations in support surfaces, and of the beams and cantilevers, not to mention the influence of image processing, such as digitizing if images are not already digital, which includes many different algorithms just touched upon in this link, in converting to grayscale, loss of resolution during resampling, brightness, contrast, and some inevitable distortion with making figures and that doesnt include the outright mistakes. So all these influences can perhaps be minimized or equalized with sample number and repetition.
3. Variation in length of the SP-D arms can be reconciled in several different ways, of which one is to normalize (which I am using) each half of the hexamer to a center point in the Ntermini joining, and an extinguishing grayscale point just after the CRD on either end of the hexamer.
4. Determining in each image the background variations in grayscale (brightness) to determine what constitutes a “background” peak not to be counted as part of the dodecamer, is pretty time consuming, and it begs the question whether the AI is better than the EYE.
5. I seems to me like finding out the background in each image is probably the most precise way to approach the variation inherent variation, and of course that can be taken to an extreme too.
6. If a grayscale of 0 – 255 is used then a significant peak would be close to 13 units in the ideal case. There are clearly times when background is twice that value. Image below shows a relatively large peak (38 or 255 grayscale) where the CRD (a portion of which appears on the right hand side of the image is just over 141.6 units. 
This image below (posted again from earlier this week) shows two examples of where the peak and valleys might be actually be reflections of underlying extraneous grit (lol). The peaks of 13 and 18 grayscale units could be debris. I guess the bottom line is sample after sample and a gradual sorting out of what is “peak” and what is not.
Measuring continuously through a dodecamer from one CRD of a hexamer to another (a node-rich line adjusted for the slighly bent shape of the arms) in terms of length show that over 116 molecules that there is no significant difference in each hexamer’s length (diameter).
137.47+11.4 (n=16) 136.23=10.47 (n=116) The t-value is -0.84948. The p-value is .198249. The result is not significant at p < .05.
All measures from AFM from Arroyo et al provides me a mean of about 135 nm from tip of CRD to the CRD top on the other side of the hexamer. Nice round number to use to measure distance in peaks.
Count, N: 349
Sum, Σx: 47177.04471
Mean, μ: 135.17777853868 + 11.09
Variance, σ2: 123.14017812959
First new measurements which will be the basis for the remaining 350+ molecules. Y axis is brightness values (0-255, x axis is nm) molecules are adjusted at the N term peak to half of the total 135nm mean from 349 measuremens just from Arroyo et al’s images.
A pitiful amount of work for one day on SP-D during this lockdown….If I had a lab and a microscope and a little SARS Cov-2 virus and some SP-D you can be sure I would be looking closely (TEM or AFM, rotary shadowed, negative stained preps .. would not matter to me) anything would be wonderful.
But just say that in different sizes of SP-D found in so many different publications, yea, even different sizes in the same publication, I am relieved that my comparison measurements (3 measurements, diameter, and length of node (cusps) in each hexadecamer) are pretty close (The one tailed t-value is -0.30. The p-value is .38. The result is not significant at p < .05) (two tailed t-value is -0.30. The p-value is .77. The result is not significant at p < .05.) they are not that different… individual molecule measured at totally different times — that is a note for consistency. There were a couple exceptions in the published bar markers where they didn’t seem to fit, and certainly the cover image of Arroyo et al did not even come close to being the size reported for SP-D requiring a 74% (or so, check previous posts for exact amount) reduction in size. (140.01 + 10.4 and 137.51 + 4.7). Some relief in terms of the method. I predict there will be a significantly greater diameter when the actual diameter is used alone, (diameter touching at least three of the 4 CRD in a dodecamer) even though it is by far the easiest calculation, i don’t think it is the best.
By adding one more molecule measure 3 different methods, two separate times the p value looks like this (The t-value is -0.21974. The p-value is .830498. The result is not significant at p < .05.)
Cover image SP-D dodecamer diameters:
Count, N: 187
Sum, Σx: 24863.56
Mean, μ: 132.9602139 + 10.79
Variance, σ2: 116.5371336
Supplemental Figure 1 duplicate of cover image diameters:
Count, N: 45
Sum, Sx: 8062.005
Mean, µ: 179.1556667 + 11.68
Variance, s2: 136.5137976
signif diff to 0.00001 cover image reduction should be 74.2%
purpose: to use the LUT plots from cover images and Sup Fig 4 i images (of which there were 15 duplicates that i used) to add data in replicate.
All together the cover and supp fig 4 measurements (3 per dodecamer) leaves the diameter at: for this AFM image.
Standard Deviation, σ: 10.416881829956
Count, N: 232
Sum, Σx: 30845.56771
Mean, μ: 132.95503323276
Variance, σ2: 108.51142705926
When i see some of these i know people are out there in this quarantine alive and doing well. Ha… Thank you to these anonymous comedians. Hopefully, Lord willing, I will have the time to assemble it into a freebie pdf. Thanks to all you humorists out there… ha ha. I just keep adding and adding.
•and perhaps the saddest laughable blooper…. Rush Limbaugh first, then and in her own inimitable way, Kellyann Conway said: “This is COVID-19, not COVID-1 folks.”
•When the quarantine is over lets not tell some people.
•Homeschooling: Anyone caught rolling their eyes or talking back will become janitor for the week.
•I thought the reason I didn’t clean the house was because I worked full-time and now that I’m home all day I found out that wasn’t the reason.
• As we enter the second week of lockdown I am thinking of Osama Bin Laden. He was stuck in the house with three wives for 5 years, and I am beginning to think that he called in the Navy Seals himself.
•Until further notice the days of the week are called Thisday, Thatday, Theotherday, Yesterday, Someday, Today and Nextday.
•How am I doing. Well, I just wiped down the container of Lysol wipes with a lysol wipe, everythings fine, everything is fine.
•Someone asks where I think I will be in 5 years. I just want to make it to April 30th.
•Every – please be safe. There is a DUI checkpoint tonight between the kitchen and the hallway.
•If they had just called this the “stay at home challenge” and posted it on facebook, the virus would be gone by now.
•Due to my isolation I finished 3 books yesterday, and believe me that is a lot of coloring.
•And just like that – having a mask, gloves, plastic covering and duct tape in your trunk is OK.
•When we come out of this and I ask you where you want to eat, don’t say “don’t know”, you had 45 days.
•Are you wishing for a long weekend still.
•Half of us are going to come out of this quarantine as amazing cooks. The other half will come out with a drinking problem.
•Anyone elses car getting 3 weeks to the gallon?
• The good news is that after home schooling my three kids for two months they have all graduated, and are ready to move out after the quarantine lifts.
•Overslept this morning, was late getting to the livingroom.
•Where is the husband – In the garden – I didn’t see him – you need to dig a little.
•The drop in gas prices during this crisis is like a bald man winning a hairbrush.
•Officer yells: Come out with your hands washed.
•No hair salons, tanning salons, nail salons: men are about to meet their spouses for the first time.
•Turns out my three hobbies are: eating in restaurants, going to nonessential businesses and touching my face.
•Grandparents missing their grandkids – when this is over you can have them for the next 6 months.
•Glad I didn’t waste my money buying a planner for 2020.
•Watching a burglar kicking in his own door I asked what he was doing – He replied “working from home”.
• Burglar, asking for unemployment.Reason for the layoff. “Everyone is home”.
•Hormel made their first batch of spam in 1937. The company announced that due to hoarding, they are going to make a second batch.
•Wear a mask inside your home to prevent overeating.
•I feel like i am 16 again, gas is cheap and I am grounded.
•I used to spin that toilet paper like I was on Wheel of Fortune. Now I turn it like I’m cracking a safe.
•I need to practice social-distancing from the refrigerator.
•Still haven’t decided where to go for Easter —– The Living Room or The Bedroom
•Public Service Announcement: every few days try your jeans on, just to make sure they fit. Pajamas will have you believe all is well in the kingdom.
•Homeschooling is going well. Two students suspended for fighting and one teacher fired for drinking on the job.
•I don’t think anyone expected that — when we changed the clocks — we’d go from Standard Time to the Twilight Zone
•This morning I saw a neighbor talking to her cat. It was obvious she thought her cat understood her. I came into my house, told my dog, … and we laughed a lot.
•So, after this quarantine, … will the producers of My 600 Pound Life just find me, … or do I find them?
• Went to this restaurant called THE KITCHEN. You have to gather all the ingredients and make your own meal. I have no clue how this place is still in business.
•My body has absorbed so much soap and disinfectant lately that when I pee, it cleans the toilet.
•Day 5 of Homeschooling: One of these little monsters called in a bomb threat.
•I’m so excited — it’s time to take out the garbage. What should I wear?
•I hope the weather is good tomorrow for my trip to Puerto Backyardia. I’m getting tired of Los Livingroom.
•Classified Ad: Single man with toilet paper seeks woman with hand sanitizer for good clean fun.
•Day 15 of Homeschooling: My child just said “I hope I don’t have the same teacher next year.” … … I’m offended.
•Better 6 feet apart than 6 feet under
•When does season 2 of 2020 start, season 1 sucks
•Where can I find toilet paper — Aisle B back
•I went to the bank teller window with a mask on and ask for money.
•How can a virus have the same name as a Mexican beer.
•The American Medical Association has weighed in on the federal government’s COVID-19 strategy:
Allergists were in favor of scratching it, but the Dermatologists advised not to make any rash moves.
Gastroenterologists had sort of a gut feeling about it, but the Neurologists thought the Administration had a lot of nerve.
Obstetricians felt certain everyone was laboring under a misconception, whereas the Ophthalmologists considered the idea short-sighted.
Pathologists yelled, “Over my dead body!” while the Pediatricians said, “Oh, grow up!”
Psychiatrists thought the whole idea was schizophrenic, whereas the Radiologists could see right through it.
Surgeons decided to wash their hands of the whole thing and the Internists claimed it would indeed be a bitter pill to swallow.
Plastic Surgeons opined that this proposal would “put a whole new face on the matter.”
Podiatrists thought it was a step forward, but the Urologists were pi**ed off at the whole idea.
Anesthesiologists thought the whole idea was a gas, and those lofty Cardiologists didn’t have the heart to say no.
In the end, the Proctologists won out — leaving the entire decision up to the a**holes in Washington
Lots of variation exists on this topic, realistically because of the variations in the properties of amino acids (this is stating the obvious). Not so obvious is the impact of the color schemes given to the amino acids, on the individual 
researchers who observe them, as they try to figure out if they have meaning. They respond to subconscious “emotions” and “thoughts” totally unrelated to science, and the colors are conveying “information” about which individuals are unaware.
Case in point, as i looked over the color scheme for amino acids from RCSB, I saw no real order or purpose. Some colors don’t even look nice, and in one case there was a duplicate (since corrected). Also since these colors are no longer what RCSB uses, here comes a new diagram.
A venn diagram described by its maker thusly: “Venn diagram grouping of amino acids based on the biochemical properties: hydrophobicity, size and polarity. Colors for the groups are derived by additive blending of the colors of representative properties.” The blending of colors to create intermediate colors could be better in my “visual” opinion, and I will have to research just how the RGB, or CMYK, or HSL, or whatever they used was calculated (original image uploaded by uploaded by Florian Battke to Research gate). I assume from the venn diagram that the following is more or less what is proposed:
Oranges = hydrophobic
Blue = small
Greens = polar
Blue-green = negative
Turquoise = small polar
Grey = small negative hydrophobic
Pink = small hydrophobic
Brown-orange = hydrophobic aromatic
Orange-pink = hydrophobic aliphatic
This swatch with letter ID is alphabetical, unlike their original which did go by color.
This approach (ref http://www.biomedcentral.com/1471-2105/13/S8/S) calls the method (integrated hierarchical aggregation tables for sequence alignment (with an unfortunate similar acronym with some group about iraq) sorts and displays
Supplemental figure 4 I is the same figure (almost) as the cover image for Arroyo
et al. I see that middle right of figure 4 becomes top middle of the cover figure, thus a 90 degree counter clock wise rotation with a horizontal flip. The figure is cropped slightly to fit the orientation of the cover but since there is no insert the former shows more molecules. The cover image is slightly squished horizontally compared to Figure 4, but I am going to assume that any manipulations of ratio of height to width would have been done in the cover not the original image (probably not a good assumption). Top figure shows the cover in near-perfect alignment with Supp Fig 4, bottom figure shows cover and Figure 4 I side be side. So measures of the cover images can be subjected to morphometry (as duplicates where they are duplicated, and as additional images where they are not previously measured using the Supp Figure 4 dodecamer bar marker. 
After a long hiatus (likely influenced by the current stay in place mandate this year) — I am posting the mean diameter of the SP-D images from the supplemental figure in the manuscript by Arroyo et al. Diameters calculated by the same two methods mentioned countless times in this blog before: diameter and segmented (node or cusp)-arm length).
Specifics are:
n=196 (three measures per dodecamer with a rare SP-D molecule with five arms, or three arms included)
Sum, Σx: 26012.577
Mean, μ: 132.7172296
Variance, σ2: 108.7097499
sd 10.426
BTW, SP-D becomes a hot topic in todays coronavirus pandemic. THough retired, I am anxious to do some work on whether SP-D might behave with this SARS Cov-2 virus as it does with SARS Cov-1
I had two sets of 3 measures each from two images in Figures 3 and 6a in the original article which I compared to identical images found in Figure 4 of the supplement. So this is a duplicate measure, two different times (half a year apart) but using the same techniques (which is to use the authors micron bar marker to determine the diameters of the SP-D molecules using two methods (1) an actual diameter measurement — where the circumference touches at least three of the four CRD in a single molecule, and 2) vector lines from tip of the CRD to the other CRD for each hexamer) sugjected to a t test which did not turn up a significant difference in the measurements made: (The t-value is -0.30062. The p-value is .769863. The result is not significant at p < .05.) That is encouraging as the values include any errors in the bar marker and any of my errors in translating the bar markers into nm in my image measurements. That give a little more credence to the methodology, however I still will compare Figure to figure and especially the cover to the other figures in that paper.) That there are differences in the size of the SP-D images from one figure image (with bar marker) and others seems likely to show up
Just working to keep my sanity. Here is a counted cross stitch of a virus particle in blues and yellows. Just ask for it or find it HERE. Be well, mm
