Fluorescent Sturgeon Spleen

[discomorphella]

This is a slide I made a few years ago from a sturgeon I caught in the Columbia river. I've posted a brightfield image previously ( http://photomacrography.net/forum/viewt ... highlight=) but I hadn't tried looking at it using epifluorescence. In keeping with my efforts to use "conventional" stains as fluorochromes (e.g. Eosin Y and Phloxine B) instead of much more toxic DNA intercalating fluorochromes and other (expensive) fluorescent dyes, here's a few shots showing what regular fluorone family dyes can do in fluorescence.
Sample info:
Acipenser transmontanus (Pacific Sturgeon) spleen, collected ~30 mins postmortem (took a bit to get to shore to clean the fish, it was a big fish), fixed in buffered formalin, dehydrated in isopropanol, embedded in polyester wax, and sectioned at 5 um using an 820 microtome (a 1935 model I fixed up). Sections were taken up on gelatin coated slides, and stained using Ehrlich's hematoxylin (blue, nuclei), Eosin Y (pink, collagen and other tissue components like muscle) and Phloxine B (bright red, granules and cytosol, especially in the erythrocytes and granulocytes). It is mounted in Omnimount2 from National Diagnostics, a really nice, safer (no xylene or other aromatic solvents) medium that has low background fluorescence (full disclosure, I know the National Diagnostics guys...).
You can see the nucleated erythrocytes, granulocytes (with red-staining round granules) and numerous other cell types in the red and white pulp of the spleen.
The fluorescence is a combination of the Eosin Y and the Phloxine B (which are chemically pretty similar) and the various chemical environments that the dyes are bound in. So the Eosin yields yellow /green fluorescence in collagen, muscle fibers and other tissue components; the Phloxine is more orange/green in the granulocyte granules and bright orange in the cytoplasm of the erythrocytes. Notice how the hematoxylin actually quenches fluorescence, so the nuclei are dark.
Optics: Leitz Orthoplan, Koehler diascopic or epifluorescence illumination using a Ploempak and H2 (broadband blue excite, > 515 nm pass) cube, NPL Fluotar 40/1.3 or Pl Apo 60/1.4 objectives. Variozoom eyepiece, Varioorthomat2 shutter module and 0.32X lens, followed by a D300. The D300 was run in bulb mode, the Vario shutter controller was used for both BF and Epifluorescence exposures. Since the D300 doesn't have an EFCS, the vibrationless (effectively) vario shutter module works well here, where I don't have anything moving (a few pics of the camera/shutter module on the orthoplan on this older post here http://www.photomacrography.net/forum/v ... highlight=).

David

[rjlittlefield]

Outstanding images and description, David -- many thanks for posting!

--Rik

[RogelioMoreno]

Very nice set!

Rogelio

[discomorphella]

Thanks Rik and Rogelio,

I should add that the light source for the fluorescence images was a regular 12V/100W halogen lamp (LH100), with no filtering except an IR cut filter. It turns out that a halogen lamp can produce enough blue light (say 450 to 470 nm or greater) to make a useful fluorescence source under some circumstances. Of course, an Hg/Xe or Xe arc source is much better, but also more expensive and difficult to obtain and use. LED sources are also good, increasingly even for UV, and also something that an amateur can build, but I thought it was interesting to show that one can get decent fluorescence images using a regular halogen source and simple fluorone dyes such as Eosin Y and Phloxine B. You could also use Fluorescein and its relatives (rhodamine, erythrosine etc) as well, although those are usually chemically bonded to antibodies for biological fluorescence work.

David

[Ecki]

Nice sections and images!

I am using a 100W halogen lamp for epifluorescence, too. It works very well indeed.