Hedgehog exo-parasites

[blepharopsis]

Here is a side view and a portrait of a hedgehog flea (confocal; autofluorescence post-peroxide bleaching. The side view is depth color-coded) and mouthparts of a tick collected from the same animal. Enjoy!

[Macro_Cosmos]

Breath-taking details there expected from confocal microscopy. I can see this winning Nikon's Smallworld contest, it's lovely! Thanks for sharing.

[Ichthyophthirius]

Really is breathtaking!

[Sumguy01]

Very very nice.
Thanks for sharing

[Tom Jones]

Wow...

[houstontx]

Amazing, what was the hardware used?

[Jacek]

[Pau]

Wow! Excellent images.

...autofluorescence post-peroxide bleaching. The side view is depth color-coded) and mouthparts of a tick collected from the same animal.

Could you explain a bit the peroxide bleaching technique and how it affects autofluorescence?
Are all the colors synthetic or are any of them original emitted color?

[Olympusman]

Very nice images.

Mike

[blepharopsis]

Amazing, what was the hardware used?

Hi, thanks! - I used Zeiss LSM 880 and a 10x NA 0.45 objective for the flea and a 25x NA 0.8 for the tick.

[blepharopsis]

Wow! Excellent images.

...autofluorescence post-peroxide bleaching. The side view is depth color-coded) and mouthparts of a tick collected from the same animal.

Could you explain a bit the peroxide bleaching technique and how it affects autofluorescence?
Are all the colors synthetic or are any of them original emitted color?

Hi,thank you for the comment. I use bleaching w/~20-25% peroxide whenever I deal with highly pigmented cuticle. Melanin is often an integral part of cuticle and probably serves as a cross-linker rendering cuticle harder (see an recent eLife publication by J. Massey "The yellow gene influences Drosophila male mating success through sex comb melanization"). Pigment poses problems for fluorescent microscopy as it absorbs bot excitatory as well as emitted light. Cuticle usually shows quite a lot of broad spectrum autofluorescence, both the color and intensity depend on the packing/organisation of chitin fibers, with highly sclerotized cuticle being brighter and green-shifted (look up papers by Jan Michels and SN Gorb on that).
The colors in the flea portrait and the tick mouthparts aren't artificially added; this is the way the microscope "sees" the sample. I used 3 laser wavelengths to excite the fluorescence (405, 488 and 560 nm) and collected the emission in 3 channels (B, G, R, respectively). After max. intensity projection you get the effect you see here (and almost always unexpected, so - extra fun!). Hope that helps!

[Adalbert]

fantastic !

[Pau]

Thank you very much for all the info, bleaching pigments now makes sense for me.

I knew the near UV/V induced chitin autofluorescence...although I wouldn't expect it with green excitation.

Getting this kind of results I can figure it being very fun!

[Robert Berdan]

The confocol microscope results in stunning images, too bad they cost several hundred thousand. I wish we could process Wide field fluorescence to look similar by combining focus stacking and deconvolution software.

Did you apply false colours or a specific LUT to create the colours or it natural autofluorescence?

When they come way donw in price I would like to own one. I suppose I could rent one at the University for about $50 to 100\hour

Great pictures

[rolsen]

I have not enough words, these are just awesome