All posts by thankusc

Image processing, signal processing? is doing both necessary?

I know well that the right brain – left brain dogma is overly popularized by the lay community, and it is easy to find discourse pro and con, but lateralization and uniqueness of each half and the terrific communication that occurs between the hemispheres is amazing, and a great area for research.
It clearly requires two hemispheres to be logical – or to be creative, and each offers valuable input, but for me, thinking in “visual” terms has become more pronounced as I have reinforced it with over the decades of microscopy. And what a wonderful evolutionary adaptation lateralization of the brain has been at providing a great exchange of perspective within a single individual’s ability to perceive what they see. This ultimately allows for inter-individual communication of ideas from those that favor one or the other approach to thought, to produce a truly global, universal “whole” mix of collective thought.
While my approach appears to be more visual, I rely on input from those that process information more numerically for help in solving problems.

Case in point is my own approach to finding out what surfactant protein D (SP-D) “looks like”, might show more neural activity in my right brain, were it mapped, while I was researching this subject.

My initial interest in SP-D, not surprisingly, came from “visual” input: albeit as an annoyance at a researcher who chose to use his “artistic licence” to produce what was an incredibly bad diagram (and to be fair, there exists a spectrum of diagrams of SP-D from the totally thoughtless to the acceptable (a couple listed here) (1, 2, 3, 4) which covered the truth that he really did not “know” in his mind’s eye what SP-D looked like even though he was researching it.

I immediately went on a quest to find every published diagram, drawing, rendering or molecular model, as well as running my own protein modeling of published sequences of SP-D on various online programs, which included those published models of the CRD and coiled coil neck on RCSB.  The search was to see if any peer-reviewed journals from surfactant research community had any models of SP-D which fit images seen under the microscope (in this case AFM, TEM (shadowing and negative staining).  None found to date.  Whats more, I found publications that totally ignored parts of the trimer, calling the CRD and neck region SP-D as if it were the “whole” of the protein, not emphasizing that it was in fact a protein that has not been completely moedled yet. The best description (as of this date 11-29-2021) there was one post on RCSB that referred to the SP-D model as a “fragment”  Kudos.

SP-D is a very interesing molecule that can multimerize, at several levels, and sometimes that organization affects function.  The models and the microscopic images provide more together than apart. I saved about 100 images from several publications (various techniques, but mostly AFM, upon which I used about a half a dozen image processing programs to ehnahce, upgrade, depixelate.  The purpose was to find a “commonality”. Those images were processed as a whole images, not just elements of the image, so I think it is/was justified. The image processing filters applied with the most successful (in my opinion) outcomes and producing the smooth and most informative grayscale plots (in my opinion) are the old standards.  Gaussian blur, unsharp mask, median, min, max (noise), and limit range.

The processed images were then assessed along a centered, segmented line trace of each arm (as the basic units of SP-D are trimeri arms) either in corelDRAW or ImageJ or Gwyddion (in which, in my experience, the latter doesnt really work here very well at all) and arm length was calculated in nm from the accompanying and simultaneously processed bar marker.  ImageJ has an easly run routine for grayscale measurements along those lines and was used to create plots exported to excel (.csv ). A screen print of the trace and resulting plot were saved with the data.  Brightness peaks were counted by eye (subjective) while the image was open in ImageJ as was peak number (subjective) counted while the grayscale plot was open in ImageJ.

Those plots were normalized over x and/or over x, y and peaks were counted again in BatchProcessing using  LTI (lag threshold and influence)(thus a semi-subjective count where any peak width of a single line width was ignored or if proximate to a bigge peak, blended), and also peaks were determined in ImageJ under the menu “find Maxima”, using three settings for “Maximum” points. These points were counted along the lines of the tracings only (ImageJ, Find Maxima; 0.5, 1, 2).

Frankly, data are all over the map. My favorite is the subjective count by eye.

My goal was to plot so many trimers that at some point the variations in the number of peaks along a plot  caused by random noise, preparation artifacts, image processing variations, publication quality, overlapping molecules, imperfect traces, etc,  would fade into a background noise that could be over come with appropriate “signal” processing of the plots and that the most likely (by some statistical measure) number of peaks along each trimer would emerge.

COMMENT: With access to a very interesting website on signal processing which defines the options for processing. and with the help of its creator I was able to learn how to use function code to assess plots (.csv plots of SP-D trimers) in Octave.  Looking over this website made me think carefully about signal processing of an image that had already been image processed….. was this in fact redoing what I had already done.  I considered the name of the algorithms being used…. they are remakrably similar, even identical names. Is this duplication….  what will be gained by signal processing my image processed signals.  (more later i hope)

Verge of a Dream: It may be

It may be because of
Simple boredom you
don’t want it
Mentioned or that
Love is like the
Weather with nothing
To be done
About it.
It may be that
Question marks
Belong to you,
Like pain that gets
Respect.
It may be
You know what’s
Been seen before
and are ready to
Toss it aside. Except
For how we hurt
Ourselves, so
Much else
is uselessly
Transparent.
Give the handsome
Gentleman a prize
And send him on
His way.
I don’t know why it seems
Necessary to make
You into words.
But a mirror cannot
show what is real
which for you is
something
That falls between
Indifference and disregard.
Amidst your thoughts
Buried like water
A hundred feet below
The surface.
There are glimpses in
Which with forbearance you’re
Now a generation like others gone
Before where
Less was given than
should’ve been gotten.

RLB 11-15-2021
notes:
Been thinking about how when someone has a bad experience, for example a loss and someone attempts to show empathy….the aggrieved says something like “no you don’t get what its like to have this happen”….”you don’t know you just pretend to understand”. “you’ll never know how it feels to lose….” etc. That conversation is in so many TV dramas, police shows (I watch British ones) or the latest was Baptiste on PBS where Baptiste is told off this way even though he had lost his daughter to a drug overdose…the other person had lost his sister….My current thinking is that saying to someone you “can’t understand” is BS..people understand in their own way and don’t have to experience in the way the grieving person experiences something. I think putting another other person down for not being as injured as I am is useless and vain anger. I was trying to wrestle with this idea in this thing I wrote….in which I am saying…a loss another feels can be understood and/or imagined. What might not be understood by someone else is how we torment ourselves, how we hurt ourselves and obviously why we do it.

CorelDRAW aggressive advertising: dont buy this software if you dont like to be contstantly bugged by popups

The more frequently you crash my CorelDRAW program with your adds dear Kohlberg Kravis Roberts of KKR, the more determinined I am to give you as much bad press as i can. How sad for you to ruin a company that was early on completely friendly with other platforms, and so kind to customers. You ruined it.  I havn’t found any fixes that work with windows 10 and corelDRAW x5.

Now that it is near black friday and the holidays, they are really bugging things, and often it causes the program i am using (two versions, CDRx5 and 19) to lose functions and then require a reboot.  Just too childish of them.    I would have been a customer for life. NOT NOW.

Totally wonderful image of SP-D

This is a screen print (originally from Arroyo et al, a dodecamer i call #51) boosted to 300ppi in photoshop, and a 3px gaussian blur added, minus-8 points in contrast, opened in gwyddion and a limit range filter applied… 100-220.  It might be my imagination, but i can see very clearly the peaks along the collagen-like domain and even details in the CRD which look so like they could be the three arms of the trimer spread apart at those ends of the molecule.  The glycosylation sites (near the junction of the N termini in this dodecamer) have texture and shape as well, possibly providing information on how many of the arms of each trimer are actually glycosylated.

There is a twisted look (as one would totally expect along the collagen like domain that is distinctly present in two of the four trimers.  Fanning out of the CRD in the lower right portion of the image is pretty amazing. In addition, the one tiny little peak that I am hoping to verify on the downslopes of the N termini combined peak is pretty nicely seen on the left middle of the center of the dodecamer.  There is a little stretching on the trimer on the left center….  moving the glycosylation portion to the CRD just a little far from the N termini group… it is possible that is why the minor peak on the downslope of that side is visible.

Also interesting in this image is the relative decrease in peaks in the collagen like domain in the area past the glycosylation peak but before the neck and CRD (where the twisty look is also evident).

Peak height and SHAPE may reflect glycosylation state

Would it be so unusual to have shape follow the number of glycosylated arms in the trimer of SP-D.  Just look at these two peak shapes.  They are certainly not the same.  Artifact from position and random events in processing can affect this i understand, but it seems to me that for sure peak width will increase with number glycosylation sites (1, 2, or 3) if in fact SP-D is glycosylated in that manner???  I have not read anything to suggest it, or to refute it.

Figure below: same image right and left, plots traced in ImageJ, the width of the glycosylation peak on the right hand trimer of the horizontally running hexamer is clearly of greater width.  RElative peak height may be indicitive of level of glycosylation as well, as it has been reported by Arroyo et al,  that the peak that is assumed to represent glycosylation is greatly diminished in de-glycosylated trimers .  Open to suggestion here.

Counting peaks along the arms of SP-D: Image and signal processing

IN this set of data there are (‘ or is’ – to use the noun as a ‘group of data’ (the latter is probably correct – “Merriam-Webster entry for “data,” notes that both singular and plural constructions are “standard” in English. and as someone posts… “anyone who ‘corrects’ you for noncount use of ‘data’ is being pedantic (and probably rude)” LOL, peak numbers along two hexamers of SP-D.

One image of a single SP-D dodecamer (which i call #51) (from a publication by Arroyo et al) was image-processed with 16 filter variations coming from a half a dozen programs,  then analyzed for “peak count” (or “LUT” peak count (aka  “grayscale 0-255 brightness”) by 2 visual and 6 different signal processing algorithms. The purpose is to determine how reliably the number of bright peaks along molecles can be counted in traditional AFM images.

Image processing does change the counts somewhat, but in all plots there are similarities in relative numbers and sizes of peaks regardless of the graphics programs and signal processing algorithms.

The two arms of this dodecamer (51) are clearly different in terms of arm length (artifact likely from stretching and twisting as fell onto the mica). The arms are significantly different in the number of peaks per hexamer.  One could think that stretching one side of the dodecamer could be useful artifact, since squishing of arms would obstruct definition between and among peaks.

Peak values were obtained as follows using the original images processed with 16 different  filters. Filter names are given below: Clearly the filters that increase contrast or mask outliers show different results.

1. Visual count (by eye – after each processing filter
2. Quick count of the peaks in the LUT plots obtained in ImageJ plots (no background subtraction)
3. Batch Process – (an app for excel files that uses dispersion peak detection to detect peaks in the LUT plots obtained in ImageJ) – (lag=1, threshold=0.5, influence=0.025)
4. Batch Process – (lag=1, threshold=0.1, influence=0.01)
5. ImageJ – Find Maxima>0.5>strict>single point
6. ImageJ – Find Maxima>1>strict>single point
7. ImageJ – Find Maxima>2>strict>single point
8. ImageJ – Find Maxima>3>strict>single point

Methods 1, 2, 5, 6, 7, and 8 use the image directly, while Batch Process uses the excel files from LUT plots.

Top figure = image processing filters

The color of the dots indicates which program was used to count peaks, and the number on the x axis corresponds to the way the image was processed before peaks were counted.

SP-D dodecamer (from Arroyo et al) above.  Arm 1 = middle left to middle right, arm 2 = top middle to bottom middle-right.

Interestingly, my eyes see more peaks than are found with image processing.  And second to that is the number of peaks that appear in the LUT plots (ImageJ) in a quick count (without subtracting background).  Summary counts or all processing (hexamer 1 and hexamer 2)  are probably pretty close to reality.

I need to find out what ImageJ uses for finding maxima … and find a good definition for Lag, Threshold and Influence.

Just an aside….. It took me an insane amount of effort to learn the programs, and to obtain and organize these data (and i need to add Octave to the list of programs)… LOL, but i think they are robust enough that using the same (or similar) scripts on numerous arms of SP-D and DMBT1 will be valuable in sorting out the LUT number of peaks per trimer.

Were I going to choose three methods to analyze additional molecules I likely would choose:  5, 6, 8, 11, and/or 16 for image processing,  then Batch Processing LTI 1, 0.5, 0.25 and ImageJ (Find Maxima 1 strict) for peak counting.

BEDE

Keeping house and yard together is a full time job, add to that my total addiction to pieces and puzzles (mosaic, quilts, stained glass, patterns etc)… and my daughter’s house and yard… all of which is falling into ruins… ha ha… but that’s oK, entropy and rebirth are part of life.

I just put my fingers on my new “pause” button, a polymer clay refrigerator magnet, and acknowledge my mortality haha.,… how funny that that piece of refrigerator art makes a difference…. i should be more intellectual and go pray, or meditate, or touch a reliqious mandala, or finger a japa mala, or kiss a talisman, or fondle worry beads…. OK now i have to look up the history of beads…… get this… “The word bead derives from the old Anglo-Saxon word “bede” (prayer). ” ha ha ha ha ha — i would never have guessed.

There is science behind this… amen.

Verge of a Dream: Heritage of Years and tears

Doubt I’m the one to ask
For what to say to your kid.
Waiting to be a kid no more.
A hard spot, for both of us.
Far from advice, not only wrong,
but ignored before forgotten.
They’ll find their own truths
The heritage of
Years and tears.
Only friends only friends
Only they
Don’t mind what’s said.
With flesh and bone
worth
So damned much more
Than gold.
To get by, you’ve got
To try
things you’re told.
And hear
how many times that
The ice you’re walking on
Is this damned thin.
Should you decide sometime
To give in and look
back, maybe what I say
Will be mostly true. There is
more luck needed than
Ever ever you get.

RLB 9-29-2021