Category Archives: Legacy Perfluorocarbons

In the 1980s artificial blood, various perfluorochemicals were being investigated as possible chemicals to use as blood substitutes. Investigations on many perfluorochemicals in the tissues of many species were part of this research plan

Perfluorophenanthrene: microscopic droplets in hepatocyte lysoso-peroxisomes

Legacy Perfluorocarbons

This is a negative I retrieved from storage (40 years past) which has a clear picture of the repartition of perfluorochemical emulsions (just some, dependent on the properties of each perfluorocarbon) within the lysososomes/perosisosomes of liver hepatocytes and interstitial cells.  This image is one I pseudocolored showing how within a membrane bound organelle, proteins coat small globules of perfluorophenanthrene (PFP – chemical image from ChemSpider, lined to name).

 

Image below neg 8946, plastic block 28373, mouse # 26, liver, 21 days after infusion with perfluorophenanthrene (i can look up the dose in mg/kg).  This type of intra-organellar, reemulsification of perfluorocarbon based blood substitute is very dependent on which perfluorochemical is infused). Please note that I have drawn around each lysosome/peroxisosome with sharpie (LOL) to define them.

Two mitochondria are present, many polysomes, and rough ER as well.  THe pink areas within mitochondria are “not identified as routine cristi, but something has changed there”.

Also note that during processing it is most likely that the actual PFP is no longer there and thus a “ghost” vacuole is present but clearly demarcated by the presence of the proteins fixed within.

 

Inner vs outer lysosomal membranes

Legacy Perfluorocarbons, Perfluorodecyl iodide

This is a quick observation perhaps meaningful (well i think it is relevant to the interaction of perfluorodecyl iodide within the lysosome). It appears to me like the inner lysosomal membrane is quite a bit thinner (meaning probably 1) a difference in the lipid membrane constituents or 2) the loss of some of the intrinsic membrane proteins.  I measured just one area of such a lysosome, and the outer lysosomal membrane seems to be about twice as thick as that which abuts the perfluorodecyl iodide crystalline inclusion.

blue perfluorodecyl iodide “footprint’, ribosome=red dot, green bar is the thickness of the inner membrane of the lysosome, dark blue is the thickness of the outer lysosomal membrane. Insert area is shown by white box, and red ribosome is shown with in the inset and the original box. liver (hepatocyte), neg 9716, block 3775, IPFD 100cc/kg (mouse) 9 months recovery

Perfluorooctylbromide: in rabbit hepatocyte

Legacy Perfluorocarbons, The cell: images, Ultimate order, the cell

Rabbit liver, two hepatocytes revealed in this electron micrograph. Three possible, one definite, inclusion left over from the infusion of perfluorocarbon, from my notes it looks like perfluorooctylbromide (19.4%PFOB 5%F68), sacrifice dates show that it was more than one year affter infusion.  The mitochondria are pseudocolored green, the microbody purplish, the PFOB inclusion complete round (the density of PFOB is greater than water, which I surmise is the reason these structural footprints are completely round and empty) and the  very tidy layer of proteins around the perfluorocarbon droplet and bounding membrane are blue.  A small (less than 100nm) droplet is typical for the remnants of infusion of the more desirable perfluoochemical blood substitutes. This particular droplet does not show a “black cap” of enzymes which often accompany the droplets. Nice little desmosome between these cells and a bile canaliculus barely seen on the right hand middle.  Golgi in the lower cell at bottom with another microbody (peroxisome – which i should have colored as well.  My recollection is that infusion of some perfluorocarbons was accompanited by proliferation of peroxisosomess (Neg 5890_block_4783_28000x.

Lysosomal granule with three visible zones

Legacy Perfluorocarbons, Perfluorodecyl iodide, Ultimate order, the cell

I have cropped three boxes from the image and changed the contrast (75Lysosomal response to the presence of perfluorodecyl iodide is kind of unique. After a 3 month hiatus (working on putative SP-A granule) it is nice to be looking at these crystal structures. I think I have posted this lysosome before, but am posting it again in reference to the three zones of texture and electron density that I am pretty sure I can see in this particular granule (not including the perfluorodecyl iodide crystals themselves). I took three areas of the crystal and changed the contrast (up 75 in photoshop) and also various levels of brightness to see whether a manipulated image would enhance any pattern present.  So beginning, and certainly an obvious “overall density” change exists in these lysosomes.  In top figure below, red dots represent noise . Below that, the differently contrasted images were vectorized using the same criteria for each so a difference in hos this renders beings more credibility to thought that there is a difference in protein density and likely protein organization in these separate crops from la single lysosome. The pattern, not yet found, just a hit that it exists.

if you are diligent, you will be able to match the textures to the exact places I cropped (yep i should have made boxes on the original so you can easily see them), but i can see the pattern in the images below and where they came from on the original top micrograph without the need for boxes.

If…Then

Electron micrographs of liver, Legacy Perfluorocarbons, Perfluorodecyl iodide, Ultimate order, the cell

I sometimes get the uneasy feeling that if i see a pattern in an electron micrograph that someone is going to say, ah thats just fixation artifact… and of course they are right. All testing is “fixation artifact” but its the purview of each observer to determine what is background noise and what is real structure.

In the case of looking at perfluorochemicals left behind in tissues, consensus says that there is “nothing” in that empty footprint, just the embedding medium (in this case EPON 812).  So there is a texture to the embedding medium, no question, but nothing I can perceive, and so easily compared to the texture of surrounding tissues, so that when I see a pattern in the tissue I am pretty convinced that it is the presence of the proteins . (They are influenced by the fixative and type and length of dehydration and other stuff not excluding the warping and shifting under the electron microscope.  All those factors play a role). It is one of the reasons that an internal control for size (and i am suggesting background texture) in electron microscopy is really important. I CAN list a short list of variables to be aware of (my knowledge is just observational) which begin even before biopsy, including the time of the last meal, 1) circadian cycle, 2) health of the tissue (in vivo or in vitro –doesn’t matter) 3) mode of analgesia or anesthesia at biopsy (paralytics – solutions with ions (as in perfusion) 4) time till tissue gets into fixative, 5) size of the tissue block, 6) type of fixative, 7) length of time in fixative, 8) length of time and chemicals used for dehydration, 9)type of plastic, 10) section thickness, 11) staining compounds, 12) TEM specs, KV, etc….  so I am willing to accept that not all parameters are under one’s control.

I will however say that the pattern below (in a portion of a macrophage in the liver of a mouse given 10%IPFD in 5%F68, 267 days post infusion) shows a linearity I would not be willing to ignore. low mag micrograph top, for orientation, next below is micrograph of the red box area which shows the periodicity. That is just one of the linear arrangements that are visible in these tissues. rotated image for easier measuring and comparing the textured are (top box) and the basic epon texture (bottom box).

Phagolysosomal and crystal patterns in IPFD inclusions in liver

Electron micrographs of liver, Legacy Perfluorocarbons, Perfluorodecyl iodide, The cell: images, Ultimate order, the cell

Two prominent areas of patterning are apparent in phagolysosomes in liver after IPFD infusion. The IPFD crystal itself, and at least four distinct protein patterns in the phagolysosomal proteins. The first image below is an enlargement of the red rectangle, and I have marked off with lines some of the pattern at the long end of the perfluorodecyl iodide crystals which seemed to work out to about 10nm in the short dimension. 23 measurements made at the end are marked off with dotted lines. A red dot=the dimension of a ribosome taken at the same magnification for reference.  Below that is the low magnification image (unretouched — I removed a scratch from the top image but no data were altered as it was not in the crystal itself). Black boxes correspond to four very distinct protein organizational patterns within a single phagolysosomal body which also included the crystal in the top image. I am pretty confident that these patterns are a clue as to which lysosomes are present, and 4 may not be the only ones which show oligomerization into ultrastructural patterns. One thing to think about is that the kupffer cells (likely) in liver acting as macrophages which engulf (and hold onto for months) IPFD crystals will have a different panel of enzymes for lysosomes (and peroxisomes) than hepatocytes, and seemingly can become multinucleated under the conditions of massive IPFD inclusions.

IPFD: phagolysosomes with protein patterned substructure

Legacy Perfluorocarbons, Perfluorodecyl iodide, The cell: images, Ultimate order, the cell

Phagolysosomes which include IPFD – 1 iodoperfluorodecane aka perfluorodecyl iodide have some interesting lysosomal enzymes.

Of these many enzymes I am thinking that there are many which are “folded” or oligomerized, that is those phagolysosomes which have IPFD show a heterogeneous substructure, zones of tube like enzymes, and areas of mostly untextured protein and then areas of unique coiled ultrastructure.  Below are two electron micrographs, the one on the left unretouched scan of a print, and the one on the right retouched (though I can hardly tell the difference – the one on the right had the coiled nature of the protein burned just a tiny bit with photoshop and several pieces of lint minimized with the bandaid tool in photoshop but no data were changed).  Red dot is ribosome (taken at about 27nm) and the thickness of one of the fibrils is something under 25nm…  which is a little larger than i measured before at 20nm but i am probably responsible for the variation), red bar is 270nm, short red lines are measurements of the diameter of the coiled (polymer or oligomer protein), pale untextured area at the top of the micrograph is the footprint of the IPFD crystal, the whole area is mostly phagolysosome. Inset is the cluster of ribosomes used for size.

 

Fluorine: wikipedia — fluoro-everywhere

Legacy Perfluorocarbons, Rants

Wikipedia’s post on “all about fluorine’– this element is so unique, and with fluorine man has made some wacko new compounds. There is only a small post on perfluorinated blood substitutes down at the bottom.  I was not amused by the fact that there were “fluorine martyrs” to fluorine chemistry experiments gone bad.  And in the old days of science, those guys really took their work seriously, without understanding (or knowing) consequences, and their lives in their hands.  I wonder if technology outpaces common sense. Of course I don’t wonder.  ha ha… it does, all the time, every day.

Finding the crystal width pattern for perfluorodecyl iodide

Legacy Perfluorocarbons, Perfluorodecyl iodide, Ultimate order, the cell

I have felt there was a pattern to the width of the perfluorodecyl iodide crystals in the micrographs that I have for some time. Today I decided to measure just two sets of crystals from two different lysosomal structures from two different cells. The graph below shows that there might be a stepwise increase to the thickness of the crystals of something on the order of 18-20nm (or maybe half that). The data from 4-8 measurements along the wide-axis of individual crystals were measured and normalized to the 27nm size of an adjacent ribosome. The mean widths sorted in ascending array and the second plot measured identically (had a few more crystals to measure) is superimposed. The red rectangles might indicate an incremental increase in size. Just thinking on this. I did find a couple of publications on “incremental pattern mining” which would be fun to examine.  Hopefully I will find someone with software already in place to do this. The actual micrographs and positions of measurement are shown below.  Red dots=27nm a presumed size for a mammalian ribosome, in each micrograph. Dont miss the lysosomal protein “snake-like protein with dark central stripe” mentioned in previous which is very clear in the top portion of the top electron micrograph. In this micrograph the values are 18-20 and 9-10 nm respectively for the two-band protein (similarly to previous post HERE).

Endosomes in phagocytes containing perfluorodecyl iodide crystals show unique substructure

Electron micrographs of liver, Legacy Perfluorocarbons, The cell: images, Ultimate order, the cell

The phagolysosomes in phagocytes which containing perfluorodecyl iodide crystals (in this case the perfluorocarbon has been retained in the body for more than 250 days – mouse liver) show huge amounts of lysosomal enzymes. The collection of macrophages (even multinucleate macrophages) don’t appear to initiate a huge collagen response, and inflammatory cells such as neutrophils, just are not that common, even when phagocytes are plentiful.

The ultrastructural characteristics of the lysosomal enzymes contents within phagolysosomes containing IPFD crystals can vary dramatically within the same cell and in adjacent lysosomes, and within the same lysosome.  This varies from a very fine homogeneous non-textured content in some areas to areas with a more coarse fibril-like texture, appearing like a lumpy (sausage link type) strand or a twisted strand (of two separate elements, or a two strand braid). In some cases the fibrils appear to be solid on cross section, but in other cases more like a tubule. This underscores the variability that would be found in a twisted fibril, allowing for the explanation of both “hollow” and “solid” profiles on cross section.

Transition zones between the types of enzyme ultrastructure are found within the same lysosome suggesting some protein overlap or continuity. There are some areas of very dense accumulation of proteins.

IPFD crystals may be large or small, and most display some obvious directionality, usually the longer side having a thin dense protein (or trilaminar membrane?) coat, but at the ends showing considerable “striping” or dragging out, or whispy periodicity.

IPFD crystals often appear in cross section to be more rounded, or even round.

Some common characteristics of the fibrillar texture of lysosomal contents of IPFD filled lysosomes follows:

  1. relatively even diameter at widest and narrowest areas
  2. often looped and tangled, folded looking
  3. equally even spacing seen as a predominant lucent area surrounding each strand
  4. often running in parallel
  5. higher mag shows sub-layering

This image below shows a very prominent (yet small in terms of nm) level of periodicity. May not be the smallest since I havn’t be able to find any information on the actual crystal chemical structure of perfluorodecyl iodide), but pretty small for conventional electron microscopy to propose, but this is so obvious that it certainly warrants comment. The single space (inset is same size as lower image and placed on an identical image, enlarged so that the lines (periodicity) could be seen.  The span is about 9-10nm line to line, and the fiber arrangement is double that…. between 19-20nm.  It appears that the protein(s) oligomerized here can come as an interconnected (maybe twisted) double strand.

Lower magnification image of the portion of a macrophage from which the inset to the right and above (white box) and the measurements of distance between fibrills (white lines)and smallest periodicity (blue box) measurements were made.  Red dots=ribosomes at an estimated 27nm for reference; blue line = smallest periodicity; purple line = the 9-10nm pair, twisted, representing the fibrills (about 20nm in thickness if section is longitudinal, or diameter ir fibrill is cross sectioned. A measurement of about 20+nm is found from center of one fibrill, perpendicularly, to the center of the adjacent fibrill, (white connected lines on the bottom micrograph show distances between fibrils measured. Image at bottom has blue line of @20+nm –the approximate diameter of the “fibril”, and the dividing line of the two strands is visible.