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Hedgehog exo-parasites
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blepharopsis



Joined: 28 Nov 2014
Posts: 148
Location: Virginia, USA

PostPosted: Thu Apr 09, 2020 11:34 am    Post subject: Hedgehog exo-parasites Reply with quote

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!







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Macro_Cosmos



Joined: 15 Jan 2018
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Location: Sydney

PostPosted: Thu Apr 09, 2020 12:42 pm    Post subject: Reply with quote

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



Joined: 07 Mar 2013
Posts: 992

PostPosted: Thu Apr 09, 2020 2:23 pm    Post subject: Reply with quote

Really is breathtaking!
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Sumguy01



Joined: 28 Jan 2013
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Location: Ketchikan Alaska USA

PostPosted: Thu Apr 09, 2020 8:19 pm    Post subject: Reply with quote

Very very nice.
Thanks for sharing
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Tom Jones



Joined: 31 Jan 2009
Posts: 299
Location: Crestline, CA

PostPosted: Thu Apr 09, 2020 8:46 pm    Post subject: Reply with quote

Wow...
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houstontx



Joined: 31 Jul 2015
Posts: 297

PostPosted: Thu Apr 09, 2020 9:30 pm    Post subject: Reply with quote

Amazing, what was the hardware used?
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Jacek



Joined: 02 Oct 2011
Posts: 5353
Location: Poland

PostPosted: Fri Apr 10, 2020 7:42 am    Post subject: Reply with quote


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https://photos.app.goo.gl/MV9IynUtBdaKkVj53
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Pau
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Joined: 20 Jan 2010
Posts: 5026
Location: Valencia, Spain

PostPosted: Fri Apr 10, 2020 8:40 am    Post subject: Re: Hedgehog exo-parasites Reply with quote

Wow! Excellent images.
blepharopsis wrote:
...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?
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Pau
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Olympusman



Joined: 15 Jan 2012
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PostPosted: Fri Apr 10, 2020 9:39 am    Post subject: Flea Reply with quote

Very nice images.

Mike
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blepharopsis



Joined: 28 Nov 2014
Posts: 148
Location: Virginia, USA

PostPosted: Fri Apr 10, 2020 11:18 am    Post subject: Reply with quote

houstontx wrote:
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.
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blepharopsis



Joined: 28 Nov 2014
Posts: 148
Location: Virginia, USA

PostPosted: Fri Apr 10, 2020 11:32 am    Post subject: Re: Hedgehog exo-parasites Reply with quote

Pau wrote:
Wow! Excellent images.
blepharopsis wrote:
...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!
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Adalbert



Joined: 30 Nov 2015
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PostPosted: Fri Apr 10, 2020 11:46 am    Post subject: Reply with quote

fantastic !
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Pau
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Joined: 20 Jan 2010
Posts: 5026
Location: Valencia, Spain

PostPosted: Fri Apr 10, 2020 11:48 am    Post subject: Reply with quote

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!
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Robert Berdan



Joined: 18 Oct 2017
Posts: 212
Location: Calgary

PostPosted: Fri Apr 10, 2020 4:14 pm    Post subject: Amazing photos Reply with quote

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 Sad

Great pictures
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rolsen



Joined: 01 May 2018
Posts: 148
Location: Finland

PostPosted: Sat Apr 11, 2020 12:05 am    Post subject: Reply with quote

I have not enough words, these are just awesome Shocked
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