Opinion: image and signal processing of micrographs

For me, signal processing offered absolutely nothing over image processing and my own reading of brightness values. It was incredible to see that Octave, Scipy, LagThresholdInfluence, and PeakDetection template smoothing programs not only ignored symmetry, but punished it. Ha. I am hoping that a “model plot” , a conscensus made from a dozen or so nice AFM images of surfactant protein D can be used to make an accurate assessment of the number of peaks along the trimers and hexamers in other images.

Verge of a Dream: Graduation Night

Some things are real.
But I never saw you
In a low back dress
On what might
have been
A street lined with
Maples, houses
Not wanting pretense.
And with the prom
I think the theme is
chiffon, You could be
My date. Except
you’ve never
Been known for
flounce or floating
around or chatting
in a circle about
the senior class
And what they’ll
Do in fall.
For all the things
I want to be real.
Nerves ignited
I never had my hand
Holding you when
They called
the slow dance.
For all the things
That could have
Been memories
So few are real.
Sitting next to you,
I can see the place
A hard bench,
A soft drink, hooking
From school, not
Single grace except
You, your brother’s
Creativity so
Brilliant I need
To turn away..or
then imagine
me a
White cane and cup.
It matters less what
Might be real,
it matters
More That when
at that turn point
that I might
Not see you when
It becomes
My graduation night.

RLB 05/30/2022

Lullaby: for Corin M Miller

Music and lyrics.

Two special souls bound together
Blended in purpose and heart
Brought forth a wondrous new child
Corin, to treasure
Blessed beyond measure
Pepsi and Bringer of Light
Tending this new precious life.

With hope and with wisdpom they’ll teach her
Mercy and grace will embrace her
Spirit of goodness abound
Love will surround
Peace will be found
Tenderly calming her strife
Guarding her new little life.

I wrote this 11 (2011) years ago for an as yet unborn Corin M Miller, who is almost 11 now. I wasn’t sure if she would ever know it existed. Technology has changed so much that a performance video can at least remind her that grandma M loves/loved her when she has her own grandkids.

Sheet music: corins lullaby-tunescribers_final

Never tell a lie: Music and lyrics by Thomas Arthur Turner

Music and lyrics to “Never tell a lie” by Thomas Arthur Turner (@1938)

Here’s a little suggestion and it might be hard to take
Thats if you’re the type who is inclined to prevaricate
It’s just a little psychology that ev’ry man should know
If he’s to be the perfect Romeo, so

Never tell a lie, here’s the reason why
Someone might believe the things you say are on the level, darling,
You might break a heart, and that’s no way to start romance.

Things you say and do, might thrill her thru and thru, but if they’re on the sly I’m telling you
You’ll make her cry, now darling, that’s no way to do, before love starts you’re through.

So never, never tell her that you love her, unless its from the bottom of your heart
For there will come a time when she’ll discover that your love for her was just a lark.

Then you’ll be a lone, by the telephone, all the girls will find
That you’ve been handing them a line, now darling,
Thats the reason why, it never pays to tell a lie.

Ridge plot, and sample plot using PeakValleyDetectionTemplate-xlsx: surfactant proteint D

The choice of what algorithms to choose for analyzing 100+ surfactant protein D images (AFM) will amount to selected image and signal processing functions. 6 – 10 plots will be made for each hexamer of a single dodecamer.  The concensus plot to which to compare all these plots will come from a concensus of peak number, and peak widths and peak heights from these data.  A composite ridge plot created from a single molecule (41_aka_45) (below) indicates that very predictable patterns of peaks and valleys do occur.  That said, it is pretty certain that there in an ideal tracing of a trimer there are 7 peaks, and something around 15 in a hexamer (which depends upon whether there are portions of two CRD in the tracing and two peaks found in N (which does happen).

1. maybe two peaks at N
2. two tiny peaks on either side of N
3. The glycosylation peak man NOT be just one peak but a set of rolling peaks – as the number of carbohydrates goes from 1, 2, to 3.
4. a consistently appearing peak peak similar in size to the glycosylation peak located on the lateral sides of the glycosylation peaks, and
two additional,smaller, as yet unnamed peaks, lateral to that peak and before the neck domain.
5. Occasional appearance of a peak related to the neck domain, dependent upon which direction the globular carbohydrate recoginition domains lie during processing.
6. One or two large peaks of similar width and height occur on either end of the hexamer (depending upon the positions the carbohydrate recognition domains fall into as they settle during processing.

Image processing:
ImageJ for tracing all plots
one image unprocessed
Photoshop 6 for sizing, dpi, contrast and gaussian blur (5px or 10px filters)
Gwyddion for gaussian blur (5px or 10px) and limit range (@100-255)
CorelDRAW x5 for graphics and normalizing plots, dividing plots into trimers,

Signal processing:
one image unprocessed (other images image processed as above)
PeakValleyDetectionTemplate-xlsx-smooth 11
Lag 5 Threshold 1 Influence 0.05 (?Stackoverflow I dont know whom to credit)
Octave, Ipeak M80
Scipy (Prominence 0.2-Distance 30-Width 5-Threshold 0-Height 0)

Data collection:
Excel and Calculator.net

Peaks will be traced as hexamers using a 1 px segmented line, always left to right, and always with an ID of which arm ( hexamers (arm 1 and arm 2), and trimers (arm 1a (always left, and 1b always right, etc). No rotation of the original image will be made (to randomize the traces in terms of possible warping in depending upon the direction of the line as it follows the molecule (this was a serious issue in Gwyddion – but i have not detected it so far in ImageJ), plots exported from ImageJ will be saved, plotted in excel. Screenprints of the molecule, tracing, and plot will be saved as “white paper”.

Ridge plot and individual plots: comparison

This is really a visual document of what I have found with a single surfactant protein D molecule (image of Arroyo et al, plots made in ImageJ, some programs for image processing include the industry standards (corelDRAW, PhotoPaint, Photoshop and others) and industry standards for signal processing (Scipy, Octave, and excel Templates (Thomas O’Haver, and others).  It demonstrates to me that the known peaks in SP-D are not the only peaks that will influence a model of the structure of that “trimer” “hexamer” “dodecamer” or other “multimer”. Each trimer shows what I believe to be at least 6 peaks on either side of the N termini junction peak (the tallest peak in the center of each of the diagrams below). The plots here are of hexamers – that is, two trimers with C term on either end of the plot (ie mirrored) , and the N termini junction in the center of two trimers.

The set of plots in the ridge plot (plots stacked and staggered, background black) are those obtained from various image and signal processing plots.  Sample plots (from one of the two hexamers of this particular protein (which i call 41-aka-45), are of arm 2). I have colored the peaks that are known  (light orange in center=N term juncture; darker orange on either ends=carbohydrate recognition domains; lighter green=glycosylation peaks; Purple peak=unknown tiny peak in the valley of N term juncture; darker green=unknown peak beaide the glycosylation peak; pink peak=consistent narrow and not tall peak; yellow=neck region, often seen peak that corresponds with the differences in obscurance by the CRD peaks as they may or may not lie over the alpha coiled neck domain.

Lower image=actual plots (some from each arm of the hexamer) to demonstrate how the different colors in the ridge plot have been determined.

Ridge plot: a single surfactant protein D dodecamer – many image processing programs

This was an idea I got from searching how to compare grayscale plots of AFM images of varioius molecules, but in this case, that is, the RIDGE PLOT. Image below is a ridge plot of of just a few of the grayscale (LUT plot) tracings made of a single SP-D dodecamer (image 41_aka_45). The variations in the plots arise from the two different hexamers (top plots are one hexamer, bottom half is the other hexamer), subjected to various image processing filters (in numerous programs). The second source of variation would be where in the center of each hexamer the segmented line for the plot is drawn (all drawn using ImageJ).  The differences in image processing filters cause noticeable differences in peak height, but change little in the number of peaks per plot.  The basic shape of the plot of each hexamer is very consistent, but the plots for the two hexamers has greater variation.

While I dont know yet if there is a way to compare the ridge plots using signal processing (I continue to look for that) in the mean time, this purely graphic representation (unedited) shows clear evidence of consistently appearing peaks between the N termini junction peak and the peaks associated with the CRD (no new news there). But the visual information is quite accurate.

Where plots are almost identical one understands that the variations in filters did very little to change the grayscale plots, where there is greater difference, then the image processing enhanced or reduced the heights of the peaks. More variability in the plots one of the two hexamers is evident (top tracings).

Image with plots normalized for x axis and center peak centered in this image.

I will use CorelDRAW to normalize the width of the plots, and center the N termini junction.

Here is a ridge plot, each tracing staggered slightly to the right, and transparent graded color marking KNOWN, and reported peaks.  Center=N termini junction, light orange; on each side of the N term, Glycosylation peaks=green; and at right and left ends, CarbohydrateRecognitionDomain peak(s) sometimes one sometimes two=orange.

Your Smile

Your Smile

Music  and lyrics by Thomas Arthur Turner

There are many thing about you
Too numerous to mention here
But the thing that made me love you-
Listen while I tell you dear

Your smile opened the door to my heart
So that I knew from the start
That you were the one for me

Your smile found I was lonesome and blue
Waiting for someone like you
A dream that you made come true

The night that we met
-could I ever forget-
I completely surrendered to you
I feel that way yet
And I’ll never regret
Even tho’ you might say we were through

Your smile lifts me to heaven above
so that I know I’m in love
In love with your smile and you.
Lyrics and music by Thomas Arthur Turner (sometime around 1938)

N termini junction peak width in nm for one surfactant protein D dodecamer, tiny peaks at valley beside it

Lots of measurements, maybe this will allow me to pick and choose which processing provides what I think is the best overall measurement of the N termini junction peak for SP-D.  This includes image processing (dozens of programs and filters and masks done separately) signal processing (several algorithms from libraries used by Octave, Scipy, some excel templates and others) and a section of citizen scientists who pointed out peak number and position.

The tiny peaks on either side of the N termini junction are elusive, and dont show up all the time. Fact is that I see them frequently but only record their widths and peaks if the signal processing programs detect them. It is likely that I pick them out when signal processing only does so occasionally As for citizen scientists (1/52 plots) does not. Therefore a comparison of that image processing vs the signal processing data will be quite different. 165/632 trimer tracings (38%) showed a tiny peak (38%).  The image processing was best at detecting those small peaks on either side of the tall N termini junction peak (detected 133 times out of the 332 plots made with images processed in various manners. Citizen scientists just did seem to see it.   The mean nm for that peak (actually those peaks, tiny, and at the valley on either side of the N termini junction peak) is shown to have a peak width (this is measured valley to valley) of 3.55nm.