The lysosomes in this alveolar macrophage from a mouse which breathed E2 for 3 hours and then allowed to recover for 5 days are awesome.  The macrophage has produced enzymes which re-emulsify the E2 into very small (in most cases) droplets within the lysosomal structure. In addition, the enzymes make a border, which is very difficult to distinguish from the trilaminar membrane which surrounds the lysosome proper.  I don’t know how to explain this look of a double membrane, but figure it is partly a physical interaction between the E2 droplet (and not unique to E2 but seen with many other perfluorochemicals) and its hydrophobicity, and slight lipophilicity.

The smallest spheres of E2 are down around 35nm diameter, and size can be compared to something just larger than a ribosome (@27nm). Picture on the top is the unretouched (i may have removed a scratch with the band-aid tool in photoshop but nothing else), and it is not too great an image (scanned from the 31/4×4 acetate negative), but interesting still, and providing lots of information. Image below is one where I have highlighted the E2 droplets in a single membrane bound lysosome and embossed them.

The dark structure (also rounded) in the upper right hand corner of the images is what I think is a phagolysosome that contains mostly phagocytosed surfactant lipids (some layering and myelin-look is seen within this structure) from the alveolar space. There are also two mitochondria near center top. It also seems likely that some of the surfactant engulfed by alveolar macrophages would find its way (during re-purposing or re-cycling) into such lysosomes containing E2 droplets.

Loooooking over an old negative of lung from a mouse that breathed oxygen bubbled liquid perfluorochemical  (in this case E2, which is a freon) I spotted what could be, maybe, seems almost possible, droplets of E2 in the endothelium (alveolar sac endothelium).  I selected portions of the image out and pseudocolored them for easy identification: Red cell within the capillary lumen, red, endothelium orange, lysosome/or vacuoles that might be E2 and comparison with those which are in a nearby alveolar macrophage, green, and the macrophage cytoplasm is purple. There are some reasonably large E2 droplets which are probably unequivocal (is that not an odd combination of words?) with more distinct membrane boundaries, and absolutely NO fuzziness within the inclusions but also having the distinctive “black cap” of lysosomal enzymes off to a little blip on some side or other of the droplet.   This is NOT seen in the endothelial inclusions…. I will look for less equivocal samples but the similarities between those and additional ones found in the macrophage is clear. 1351_4840_mouse_E2 breathing for 3 hours, 48hr recovery, capillary_endothelium and two macrophages.

Looking at lysosomes in the lung of mice which have breathed perfluorochemical (PFC) liquids is interesting. These are old archived micrographs from the early 1970s but have a lot of valuable information about the reactivity of perfluorochemicals in the body (the latter being touted as totally inert.  Regardless of what science says about the reactivity of PFC they do cause a mountain of effects in humans, not the least of which is an immediate uptake by the reticuloendothelial system, massive sequestering and dumping of enzymes into the resultant lysosomes, and a kind of partitioning, or re-partitioning, or re-emulsification of the PFC, depending upon whether it was breated as a neat liquid, or infused as an emulsion with other agents accompanying it.

So “case in point” is this alveolar macrophage, picture taken from the lung of a mouse which breathed E2 for 3 hours and was allowed to recover for 48 hours then lungs taken for electron microscopy (and probably chemical analysis, though I have no clue where those data are at this point 40 years down the pike).  I just know that my negative number is 1351, and that block number is 4840, magnification was 5,000 (Zeiss 10) date 2 17 1975 at 60kv.  Mouse weighed 16.85g, breathed E2 for 3 hours on 2 3 1975, and was processed on 2 5 1975, which equates to 48 hour recovery. The sample of lung was taken from the R, mid lobe.

The image below (L) is a portion of the original negative (scanned as a transparency from the film) with an enlargement (box demarcates it) on the right where I have “pseudocolored” the protein densities with purple, which surround the empty footprints where the PFC was fixed (leaving the nice round spaces) in the cytoplasm. About 6 ribosomes (@27nm diameter) are also highlighted beneath the central phagolysosome?lysosome?membrane structure to show that the size of the smallest perfluorochemical droplets is in the nanometer range, before — before what, perhaps being small enough to diffuse back into the alveolar air space and be exhaled.  I don’t believe anyone knows whether the larger or the smaller E2 droplets are more apt to move into the alveolar air space.  There is one tiny droplet which is about 27 nm nesteled between two ribosomes… go figure.  The enzyme response here is pretty amazing, and appears in section to be most often seen as spheres, but is more likely to be a longer sausage shaped lumpy body.

The bottom line of this image is that these droplets appear to migrate along the membrane systems.

Alveolar macrophage from a mouse lung, the latter having been subjected to complete submersion in E2 perfluorochemical for three hours, and allowed to recover for 48 hours.

A single macrophage has almost all the PFC inclusions with high concentrations of enzymes, making them appear to have black “caps” in some cases, and in others, mostly enzymes, showing the tiny E2 particles and larger particles (does anyone know if this is Oswald ripening…. it seems as if the particles are doing two things, getting smaller and coalescing…??). The variety of multivesicular bodies here is quite amazing, with some having other structures included, some being filamentous, mostly being very very electron dense.  Each of the MVB/LE with PFC inclusions (i did not tag them all, it was too cluttered) have a bounding box indicated on the unretouched first image,  they are all enlarged and collected in a gallery below (numbered so you can find them).  Compare the variety here with three other posts on MVB/LE/PFC.