Amoeba fluorescence

[Ecki]

I have added a 100W halogen lamp for simple epi-fluorescence applications to my setup. I have used a filter combination that allows for 450-490 nm excitation and and blocks emission < 515 nm. Or, as I had to explain it to my daughter, blue light hits the specimen and blue light is blocked from the observation. All we see is fluorescence.

I created a very low concentrated Basic Orange solution (1:10000). Basic Organge is a fluorochrome that can be used for in-vivo staining. The nuclei will show green fluorescence, cytoplasm green fluorescence and lysosomes orange fluorescence.

A drop of the Basic Organge solution was added to the object carrier.


63x, 450-490 nm excitation filter, 515 nm emission filter, Basic Orange

We see some algae being digested. The freshly captured ones still show red chlorophyll autofluorescence. The bright green spot is the nucleus. Around the older digested algae we see some orange spots - maybe lysosomes. What the small green spots are I don't know.

Best regards
Ecki

[tpe]

great to see so much and so many different things. It must be very tricky getting a reasonable amount of light from the sample, and would be interesting to know what fraction of the light is returned through the flourescence. Thanks for the explanation too.

Tim

[rjlittlefield]

Ecki, this is very interesting. I have no experience with these stains and it is great to see what they can make visible.

But one thing -- I noticed this image is pretty dark, so I pulled it into Photoshop to brighten it up and see if I was missing something. It turned out that I was missing a lot! Adding a Photoshop Levels Adjustment Layer with parameters [0,1.15,189] exposed much internal structure that I did not see in the original image.

I would like to get your thoughts about the change. Can you try it yourself? Or if you would like, I could send you the brighter version or post it out.

--Rik

[Ecki]

Rik,

Or if you would like, I could send you the brighter version or post it out.

Yes, please post it!

I must admit that I am photoshop illiterate.

regards
Ecki

[rjlittlefield]

Ecki, here is the brighter picture and the way I made it, by adding a Levels adjustment layer. I have circled the two sliders that I moved to adjust brightness and contrast.

--Rik



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[g4lab]

If anybody is interested in venturing into the fluorescence arena I probably have more gear of that sort than I can ever use which I collected for other purposes. If anyone wants help send me a PM.

[Ecki]

Rik,

thanks for posting that image. Yes, it looks better that way. Normally I don't use PS at all as I am unsure whether the image is better or worse after my dilettantish operations.

Regards
Ecki

[PauloM]

If anybody is interested in venturing into the fluorescence arena I probably have more gear of that sort than I can ever use which I collected for other purposes. If anyone wants help send me a PM.

Hello Gene,

Have any spare gear that would fit my Nikon -phots?

One thing I am looking for (and which I have little hope of finding) it a spare part for an older Nikon EF-D illuminator, back when the lamphouse screwed into the illuminator. More recent models have a bayonet mount, but my unit is the screw-in type, and the threaded part of the illuminator is broken.
See the picture, with the fracture marks:


Until I get that sorted out, I can't start using that illuminator, and because of the weight that it will have to stand, and the high temperature it will reach, I have no idea what I can use to glue it back together.

Other than that, i'm always on the lookout for upgrades (to a Quadfluor unit, for instance) and for cubes.

Cheers,
Paulo