Fast green staining of Swabs for the large collection of data and cross referencing.

I am reviewing observations I haven't shared and checking them again with vector based LLM to give me refinement. Extremely large project, but essential and fun.

Read my blab before at the bottom before assuming this is a good idea for everyone to be trying, you’ll see why. But it cant hurt to see what i got from my sample REVIEW this time by the VECTOR BASED LLM which had other instructions, not just imagery. This is my work on swabs having done another round of Fast Green staining.

Detailed Observations of Rapidly Formed Structures in the Sample

Key Morphological Observations

Crystalline and Irregular Structures

1. Highly Refractive, Angular Crystalline Structures:

   • Found prominently in the first and second rows.

   • Clear edges with birefringent properties under brightfield microscopy.

   • Indicates crystal formation, possibly salts, protein crystallization, or lipid-crystal phases.

   • Irregularly shaped fragments may suggest brittle crystalline materials.

2. Larger, Polychromatic or Aggregated Crystals:

   • Some crystals show hints of internal heterogeneity (color variations), possibly multilayered or composite materials.

   • May indicate protein-salt complexes or materials undergoing precipitation.

3. Needle-Like and Fiber-Like Structures:

   • Long, thin structures observed in the second row (middle image and bottom images).

   • Possible interpretations include:

     • Protein fibrils: Suggestive of rapid protein self-assembly.

     • Salt crystals: Needle-like habits are common in certain salts (e.g., sulfates, chlorides).

     • Hydrogel fragments: Stained portions suggest dye binding.

Blue-Stained Branching Features

1. Starburst Patterns:

   • Distinct radial structures appearing in some areas, notably in the last row.

   • Suggests proteinaceous or polymeric assembly into organized geometries.

   • Often associated with hydrogel networks or polysaccharides interacting with proteins.

2. Filamentous or Fragmented Networks:

   • Fine, thread-like structures in the last rows and certain aggregated regions.

   • Points to rapid polymerization, possibly proteins, polysaccharides, or fibrin-like assemblies.

Dye Distribution and Staining Patterns

1. Localized High Dye Affinity:

   • Strong staining in crystal-like and networked regions, indicative of:

     • Proteins rich in charged groups (e.g., amines or hydroxyls).

     • Salt-laden matrices with trapped proteinaceous material.

2. Gradient Staining Patterns:

   • Some areas have faint staining, possibly suggesting areas of less protein or dye penetration into denser material.

Dynamic Insights into Material Formation

Formation Dynamics

1. Rapid Phase Separation:

   • The mixture appears to undergo quick phase separation, with denser crystals forming alongside dispersed networks.

   • Indicates instability or rapid precipitation upon mixing.

2. Role of Environmental Factors:

   • Crystallization and needle-like structures suggest ionic interactions, likely influenced by pH or ionic strength.

   • Blue-stained polymers point to secondary processes, such as crosslinking or hydrogel network formation.

Potential Mechanisms

1. Protein Crystallization:

   • Fast Green binds strongly to proteins, and their rapid assembly into organized crystals is likely.

   • Proteins may precipitate due to oversaturation or interaction with ions.

2. Polysaccharide-Protein Interactions:

   • The branched, filamentous, and starburst patterns suggest organized co-assembly with anionic polysaccharides (if present).

   • Such coacervates form under ionic stress or mixing with multivalent ions.

3. Salt-Protein Aggregation:

   • Crystalline forms with layered structures are typical of salts interacting with charged residues in proteins or other amphiphilic molecules.

Relevance of Observations

1. Evidence of Rapid Precipitation and Structural Differentiation:

   • These rapidly formed structures highlight significant instabilities in the mixture, leading to quick phase separation.

   • Crystals, fibrils, and networks may represent distinct materials (proteins, salts, or polymers) forming independently or through interactions.

2. Distinct Features Indicating Material Properties:

   • Crystalline structures: Point toward salt or protein crystal formation, valuable for studying ionic interactions or crystallization dynamics.

   • Branching patterns: Suggest hydrogel-like networks forming spontaneously, offering insights into polymer-protein systems.

   • Needle-like features: Indicate conditions conducive to anisotropic growth, possibly linked to specific ionic or molecular templates.

Conclusion

1. Primary Materials Likely Present:

   • Proteins (dominant candidate, forming crystals, fibers, and networks).

   • Salts or ionic complexes (forming crystals and influencing phase separation).

   • Polysaccharides or hydrogel precursors (branched or filamentous networks).

2. Key Implications:

   • These rapidly formed structures are indicative of rapid ionic, pH-driven, or concentration-driven precipitation dynamics.

   • Staining reveals distinct protein, salt, and potentially polysaccharide contributions.

Detailed Observations from the Fast Green-Stained Sample Images

Key Observations

Microscopic Features

1. General Features Across All Images:

   • Highly varied structures, including spherical, lamellar, irregular, and crystalline regions.

   • Fast Green staining shows intense coloring in specific regions, indicating the presence of proteinaceous material, particularly those rich in amine or hydroxyl groups (often associated with proteins, peptides, or certain lipid conjugates).

2. Morphological Structures Observed:

   • Spherical Structures:

     • Observed in many images (e.g., droplets in both large and small forms).

     • Likely candidates: Lipid droplets, vesicles, or emulsified particles.

   • Budding or Fragmented Structures:

     • Small particles or droplets budding from larger ones (e.g., in bottom-left sections of some images).

     • Possible interpretation: Coalescing emulsions or vesicle aggregation.

   • Lamellar or Layered Patterns:

     • Lamellae visible in regions with concentric structures, indicative of multilamellar lipid vesicles or protein-layered complexes.

   • Crystalline or Polygonal Regions:

     • Irregular edges and crystallized structures in certain zones.

     • Likely composed of salts, lipid-crystal phases, or protein precipitates.

   • Emulsified Areas:

     • Presence of dispersed microdroplets surrounded by larger domains suggests phase separation (common in emulsions).

   • Boundary Staining:

     • Strong staining at droplet edges and some interfaces suggests surface-active materials (e.g., amphiphilic molecules such as phospholipids or protein-lipid interactions).

Magnification-Based Observations

1. 25X Magnification:

   • Broader overview of large-scale coalescing structures.

   • Droplets ranging from tens to hundreds of microns.

   • Evidence of irregular particle aggregation at boundaries.

2. 40X Magnification:

   • Detailed views of microdroplets and lamellar layers.

   • Enhanced visualization of thin structures and inner domains (e.g., possible multilayers or thin films).

Candidate Interpretations

Material Candidates

1. Proteins (Primary Candidate):

   • Evidence:

     • Fast Green selectively binds proteins (amines, hydroxyl groups), showing intense coloration in spherical droplets and lamellar regions.

     • Morphological resemblance to protein-rich emulsions or coacervates.

   • Potential Types:

     • Protein aggregates (e.g., casein micelles, gelatin droplets).

     • Protein-polysaccharide coacervates (if carbohydrates are present but unstained).

2. Lipid-Protein Complexes:

   • Evidence:

     • Spherical domains and lamellar structures could correspond to lipid-protein complexes.

     • Fast Green may stain protein-associated phospholipids or surface-active proteins at droplet boundaries.

   • Potential Types:

     • Liposomes stabilized with proteins.

     • Emulsions containing amphiphilic proteins (e.g., emulsifying agents like lecithin or polysorbates with protein interactions).

3. Lipid Vesicles (Possible but Secondary):

   • Evidence:

     • Multilamellar or unilamellar patterns resemble lipid vesicles.

     • Boundary staining suggests lipid bilayer structures.

   • Potential Types:

     • Multilamellar vesicles (if lipids dominate).

     • Phospholipid emulsions with protein residues.

Role of Crystalline Structures

• Likely salt inclusions or precipitated proteins, as Fast Green does not stain crystalline lipids but may interact with ionic residues.

• Could also represent residual by-products of preparation.

Concluding Observations

1. The primary materials appear to be proteinaceous, potentially forming aggregates, coacervates, or emulsified domains.

1. Lipid involvement is strongly suggested, with vesicles or emulsions possibly stabilized by proteins or surfactants.

2. Crystallized regions likely represent salts or precipitated residues of proteins or lipids.

1. The lamellar structures are consistent with multilamellar vesicles or layered protein complexes.

Further verification could involve specific experimental methods:

• Confirm protein presence via spectroscopic analysis (e.g., UV/Vis or FTIR).

Use additional dyes specific to lipids or carbohydrates to verify secondary components.

I shan’t make this much longer by re-verbalizing a summery of what is above since the LLM did that quite well. You can see what it summarized as possibilities and it even gave me a percentage of accuracy figure against its self based on its own interpretations matching to literature, also not a trustworthy figure in all cases. The results are quite telling and does highlight what is to be expected from dye adhering to certain structures which would look and behave like these. I am very, very certain still to this day that most of the work so far has shown at least 4 types of polymer that can easily be seen by uptake in solid structures which resist particular dyes and not others.

pollysaccharides and certain other lipid materials are on my probability list for further analytical investigation. Lists of Bio-compatible hydrogel polymers were collected some time ago and I did stain samples for many of these and a few possible positives.

If i put a large amount of data in the LLM via the separate VECTOR channel it seems to conclude many of the findings I have made over the last 2 years and sometimes suggest something new as a possible detail. So I am quite pleased with what I have learnt so far studying these horrid stuff and that LLM’s recent use has indicated a reasonable accuracy on structures or materials as I believe them to be. Still don’t know enough though, its a witches brew as friend says. Recent science studies like that by Dr Speiker, Dr Young Mi Lee, Dr Arnold Burkhardt, and others have enlightened us all to the kinds of protein complexities, Constructive DNA, and other material unanimous with the kinds of materials being seen here in my studies. Self-assembling, trans-humanizing, hell on earth. No robots seen yet by anyone via SEM or other methods. But this stuff is all you need to make wireless reorganization of people according to many studies. It is way out of specification for that of anything medical and shows signs of complex bodily reorganization as an intent.

I added a few dark field stained images to the LLM vector and had it try to see any colour shifts it could compare and correlate. That is even more dangerous an idea since many or most dyes are not used via dark field enough to support interpretation via reference of fact at all. A few are known. Some dyes like Congo red can be used to stain amyloid structures of some types, which can then be confirmed by cross referencing to a polarized microscopic view of the same sample.

Further experimental characterization (e.g., spectroscopy, elemental analysis) is recommended to confirm the composition and interactions.I don’t have time to add all the info and references. This is not refined information and LLM even with a vector is not conclusive at all. It has made loads of mistakes before. So when I see new interpretations each one has to be queried, read up on, and other possibilities or interactions have to be considered. That is where some basic to mid-level knowledge of dyes, their interactions with material, and possible interaction issues or throw offs are needed at least. Usually when I factor in other dyes and techniques it shall change its mind on some things it previously had concluded.

When I originally started using dyes on this material a couple of years ago I had to start from scratch using what I had learnt from staining biological samples and fungal spores whilst trying to investigate disease and look at other cool stuff as people do. So I know it isn’t as easy as the LLM will make it seem. Dye use is actually very complicated, but the LLM can help save time and offer further confirmation if you know how to command instructions to it as a primer with conditions and boundaries based on some knowledge regarding dyes and the traps using them. Dyes are known as indicators for a reason and can give false positives if not mixed right, store right, used correctly, and even when they are can still be wrong on some occasions.

I agree with much of the LLM’s more obvious statements here regarding polymers and polysaccharides, but have to review deeply some of its observations for which myself had not made to that level previously. It is kind of fun for sure though, and opens new avenues fine details to be explored.

I had someone email me a while back who bravely tried to replicate my last post using similar techniques with the LLM and they got entirely weird results appear which did not make sense or even correlate with what I had been concluded. It is not easy to explain how I added to its vector instructions since if you are not aware of certain things in the first place regarding this material then you cant stop it making wrong assertions, or entirely wrong observations. I had to laugh to myself because it was great someone tried to bravely go at it, but the results were funny considering and the traps highlighted themselves right there.

If your sample comes out as cat food you can be sure it is wrong. :D

How do you know if it just sounds sciencey and the believe the LLM is correct? That is the danger of it, and it is bound to happen a lot. Anyway, still….. imagine all the simpler stuff and clues it could give some guys researching.

All this said, I would not advise anyone to ask it questions and presume that the info is correct at all, unless you have some experience and can tell if its being a cheesy liar. You will have to go deep down to every detail, reading studies for some time if you do not have background in using dyes and experience of certain material caveats. Worth doing for many reasons, or just for fun! The more you know, is the more know, and the more fun it is to stick stuff under the scope while being able to interpret something new.

I actually find it very hard learning new dyes with unique material because the literature is not always great and sometimes there is nothing out there to explain why the structure or thing you are dying is that colour, or if it is even what should be expected to happen. I am no master with dyes and most of my experience was using about 15 dyes such as Hucker gram stain for parasitology, methylene blue, safranin, crystal violet, India pink for spores, cotton phenol blue for some fungus, etc and so on. I use them cautious and with people to review my interpretations when i use them for serious stuff like this. I am usually on key with it, but it doesn’t hurt to have help and confirmation when it involves critical observation.

I had a problem when our chemistry scientist friend and a sciences teacher were trying to help me find out why Toluidine blue was dyeing certain structures in some colours on the swabs over zoom scoping last year. for this particular material there were no clues, none of us could find references past 3 minor observations that were not conclusive of anything and so had to ignore any indications from using the dye except for DNA and RNA. The LLM did pull up some info on it, but we couldn’t assume it was trustworthy since it could not provide references to papers for us to examine and so must have been trying to make conclusions based on the LLM’s understanding of the chemistry at hand (room for tonnes of error there). It could have been right, but can often be wrong, and interpretations like that must come from studies unless we want to reinvent the wheel sort of and trip up, most dyes and their specific uses are well known in literature and in daily use. Toluidine blue has amazing interactions with tonnes of materials, looks really pretty in bright and dark field, but few know how to interpret that one past staining for RNA and DNA which it does very well. Anything that comes up in other colours, and it will do that, is too difficult to interpret by guessing. However i have used that to detect DNA and RNA possibilities in many of these samples. I have to do it again though to be honest, for consistency.

I have new dyes on my order list to be sent here. They are not your average dyes and can only be obtained from pharma companies at a hefty price. Let me know if anyone can send dyes guys, I can pay, just cant get access for some. Fluorescence dyes would be nice at one point too since I have the ability to view samples that way too. DAPI has broad uses and can also be used for some synthetic material analysis which is backed by studies galore.

But at the end of the day, dyes or stains are just for clues as INDICATORS. My intention is to use this data towards conformation and as backing for spectrometry and other methods which are tangible. add all this work together and correlate it to get a very accurate and tangible outcome. One science accepts and scientist will look at deeply for further investigation.

DON’T FORGET , your help is appreciated in getting help purchasing the LCMS (LIQUID CHROMATOGRAPHY MASS SPECTROMETER). I have my finger on the trigger but worry about buying an un-refurbished model cheaper. I don’t really have time to be pulling it apart to replace broken detectors or faulty pumps and my smart mate who used to service them doesn’t want to catch a plane to help me fix one either :D. just kidding mate.

It is best to get a checked refurbished and confirmed unit used.

Thank you all for supporting and reading. I wish everyone the best of everything. Good night Y’all ! :D