Category Archives: Perfluorodecyl iodide

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

Iodoperfluorodecane (perfluorodecyl iodide)

Perfluorodecyl iodide

Previous comments about some kind of order – periodicity – linearity? in the micrographs of hepatocytes which contain inclusions of perfluorodecyliodide (IPD   )  and there are others that I noted. (nb. quote)Ubiquitous, highly glycosylated, integral membrane proteins of largely unknown function, called lysosome-associated membrane proteins (LAMPS) or lysosomal integral membrane proteins (LIMPS), account for about 50% of the protein in the lysosomal membrane.

1. the trilaminar membrane has an exaggerated density on the outer membrane, and a very smooth (maybe no integral or surface proteins) on the lamina of the membrane in contact with the IPFD. See picture below, blue arrow inner trilaminar membrane (maybe few proteins) outer trilaminar membrane (red arrow) with maybe lots of membrane proteins. It would be interesting to nkow for sure if the IPFD causes this lack.

Another interesting thing is that there is a substructure to the lysosomal protein matrix — in fact at least three different organizations that I can detect, and this one just happens to match up in terms of size with LAMP-1…. for which there are shadowed images by Carlsson and Fukuda of the dimer found here which i have sized using their micron bar marker and the size of a ribosome (used 25nm) and superimposed over a giant lysosome found in the liver of a mouse that had been infused with IPFD (perfluorodecyl iodide). (see below) Tiny red box (enlarged in inset) is their image of the rotary shadowed dimer of LAMP-1, which i have placed into the textured density of one portion of a large lysosome containing IPFD crystals.  Red dot is an aproximate size of an hepatocyte ribosome (25nm used).  Wouldn’t that be a fun thing to spend some time figuring out, since I have only suggested it, not verified it.

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.

Wedding cake crystals: perfluorodecyl iodide

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

These crystals are certainly odd. I have for many years wondered at their layered cake appearance, and today just found it so funny that i had to make a spoof about it. So here are a few images taken from within a single hepatocyte phagocytic cell (like a resident macrophage) and these layered cakes ‘so to speak’ are oriented with the largest layer at the base and stacked vertically. The crystals appear to be stacked round layers, but probably not always, maybe with a chance orientation of smaller layers “up one”, I have not done any counting to see whether they might be “reducing in size as they stack”. The rounded bottom layer (not shown in these images) is seen in cross section sometimes as a full circle, other times as a tangential cut with one straighter edge and one rounded edge, just as one would find in a tangential cut of a layer cake.

At any rate, these are clues about the structure of perfluorodecyl iodide crystals in vivo.
intracellular perfluorodecyl iodide crystals stacks of discs

Spleen of a mouse receiving perfluorodecyl iodide emulsion–

Perfluorodecyl iodide, Ultimate order, the cell

The spleen, an immune organ, initiates immune reactions to blood-borne antigens and filtering out foreign material and spent circulating cells. It comprises at least 2 primary cellular compartments: the white pulp with its “periarteriolar lymphatic sheath, marginal zone and follicle” and the red pulp with its “reticular fibers, reticular cells, and associated macrophages”. These compartments have different vascular and cellular architecture.

Looking at mouse spleen after the infusion of a 10% IPFD 5% F68 emulsion at 100CC/kg, 55 days post, I had expected spleen to be completely packed with macrophages full of IPFD particles and yet I dont think (this is an estimate) that the volume density of crystals would be any more than 1-3% of the whole spleen.  It is also clear that white pulp is pretty much devoid of cells with IPFD crystals, and IPFD crystals were pretty much limited to the red pulp. A quick look says that megakaryocytes don’t take up IPFD, red cell production, mitotic cells dont have IPFD inclusions (or much of it) that areas that are most likely to have cells with IPFD inclusions are around blood vessels.  I have spleen tissue from infusion of many other prerfluorochemical emulsions…. spleens with IPFD are going to have to go to the back burner till I can figure out some fun stuff about liver.

It looks like IPFD does exit the liver after a period of 200 days or so, the crystals in the liver macrophages have largely disappeared, but what is left in the wake of the crystals is a host of cells that are in the interstitial and periportal spaces which should not really be there. The macrophages that were multinuclear remain that way with many dark areas (organelles) likely remnants of the massive lysosomal structures that were present during the IPFD occupation.

Perfluorodecyl iodide crystals in vivo: big

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

Just reviewing some slides of liver of mice given infusions of an emulsion of perfluorodecyl iodide….  some of these crystals which completely fill to overflowing hepatocyte macrophages (multinucleated macrophages or macrophages fused into multinucleated giant cells following the foreign body response to IPFD infusion). Just saying here, some of the IPFD crystals are upwards of 30 microns long, and vary in width from 10nm to many microns. There is a very obvious directionality, always longer than wide. Iin terms of the actual molecular polymerization of IPFD, which is necessary to establish what is “wide” and what is “long” — that remains to be determined. But for now, these macrophages are about 90% “other” with half dozen nuclei in a localized cytoplasmic – crystal free zone together.

And regarding spleen, it has far fewer cells containing IPFD than I would have expected (just a relatively small number of reticuloendothelial cells show collections of crystals (compared to liver from the same animal which does have multinucleated cells full of crystals). So that is kind of interesting.

55 days after 100cc/kg of a 10% IPFD 5%F68 emulsion, the hepatocytes themselves are very well protected from inclusions of IPFD…. there are some probably, but not many. Hepatocytes themselves look nice, but the clusters of macrophages, without eliciting much of a fibrous tissue response are neatly circumscribed.

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.

 

Peroxisome in mouse liver after perfluorodecyl iodide

Perfluorodecyl iodide, Ultimate order, the cell

Peroxisomes: I have learned a few basics about these organelles. They are an old organelle evolutionarily, and the enzymes within have become unique to the organisms, species, and cell types wherein they function.  So in the case of the liver of the mouse, there are going to be many enzymes, but basically for the following purposes: 1) for directing peroxiome-relevant proteins produced by the RER into two places, 1) including the peroxisomal limiting membrane, and 2) the peroxisomal matrix.  Matrix enzymes would include those that are protective, in that they metabolize reactive oxygen molecules, and participate in fatty acid metabolism, and peroxisomal matrix those enzymes that have evolved with the species and its environmental pressures and xenobiotics (which would