I suggest optimizing the specimen preparation and imaging conditions before final optimization of the image analysis.
Can you prepare a more dilute preparation with less agglomeration? For colloidal particles we used 5x5 mm Si chips Ted Pella #16008 plasma-ashed in air (in our 1980 vintage “standard” plasma asher) or - later - in our Gatan Solarus plasma asher with a H2/O2 gas mixture. This treatment produced a wetable surface for deposition from a suitable solvent.
Getting colloidal particles well-dispersed is often challenging even when the solvent wets the substrate. We had some success with a glass nebulizer
Ted Pella #14601. There are now commercial electrospray devices that do a great job; I wanted one but the lab could not afford it… Our best dispersion came from depositing on ashed Si chips in a Beckman Airfuge.
Once the prep is optimized, perhaps consider recording images with a higher magnification so the smaller grains are better sampled in the image. Depending upon the polydispersity of the particle area you may need to record images from many fields to get reliable statistics. We typically included the
Shape descriptors in the
Set Measurement dialog.
I’d like to share with you something my Ph. D. advisor, Prof. E. L. Thomas, taught me from my first day: That I should be the biggest skeptic regarding my image/data analysis. One of the first documents he gave me was an excerpt from Henry Baker’s book “The Microscope Made Easy” (1743). I suspect that Baker learned this the hard way. I sure did on a couple of occasions when I ignored his advice…
The quote below comes from pages 62 and 63. I changed the case of some words for modern English usage.
Beware of determining and declaring your opinion suddenly on any object; for imagination often gets the start of judgment and makes people believe they see things which better observations will convince them could not possibly be seen: therefore assert nothing til after repeated experiments and examinations in all lights and all positions.
When you employ the microscope, shake off all prejudice, nor harbor any favorite opinions; for, if you do, 'tis not unlikely fancy will betray you into error, and make you see what you wish to see.
Remember, that truth alone is the matter you are in search after; and if you have been mistaken, let not vanity seduce you to persist in your mistake.
Pass no judgment upon things over-extended by force or contracted by dryness or in any manner out of their natural state without making suitable allowances.