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Cartilage
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Pitufo



Joined: 21 Jun 2015
Posts: 171
Location: United Kingdom

PostPosted: Fri Feb 09, 2018 10:43 am    Post subject: Cartilage Reply with quote

I managed to pick up a used ET DAPI GREEN FITC filter cube yesterday at a reasable price, so thought I would give it a go on an old DMRB.

Excitation is in the violet and late blue regions, so rather than rig up a mercury light source I thought would just try to switch the 100W halogen lamp housing onto to the epi port.

A pleasing blue light shone down from the objective and after trying a few slides I found this hyaline rabbit cartilage which produced bright autofluorescence.

20x Fluotar objective with Touptek cam. Stack of 25 images.


Cross-eyed stereo


These are the specs of the cube. If anyone has suggestions of what else would make good a good subject, I would be interested.

This cube seems suitable for observing DNA staining with acridine orange (which is cheap, reasonably safe apparently and actually produces a green emission with DNA).

https://www.chroma.com/products/sets/59001-et-dapi-fitc

I was considering removing the 59001m emission filter to get a better output range for autofl but is neatly glued in place in the same frame as the bs mirror. The 59001x (excitation filter) can be quite easily removed and then I could add a mercury light for some UV excitation.


Last edited by Pitufo on Fri Feb 09, 2018 11:33 am; edited 4 times in total
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micro_pix



Joined: 11 May 2012
Posts: 141

PostPosted: Fri Feb 09, 2018 11:04 am    Post subject: Reply with quote

What a great result! Super image.
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Pau
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Joined: 20 Jan 2010
Posts: 4275
Location: Valencia, Spain

PostPosted: Fri Feb 09, 2018 11:58 am    Post subject: Reply with quote

Very nice!
Actually I can't see any stereo effect although the single image is pretty 3D itself
Congrats, these multiband filters are usually very expensive
Quote:
These are the specs of the cube. If anyone has suggestions of what else to look at I would be interested.

Not sure about what are you asking
- about the excitation light, this will work very well for UV and V excitation producing both blue and green fluorescence. UV LEDs are now enough powered to avoid the need of mercury lamps, Nichia, LG and LedEngin have adequate models in the 380 - 400nm range.
Long blue (not "royal blue") and many white LEDs would also be good excitation sources for your filter set.

- about subjects, with UV you will find lots of autofluorescence in plants, arthropods and fungi. Pollen of some species, molds and fern sporangia will produce nice emission with blue excitation

If the filter cube is from Chroma it can be dismounted, although it is so well matched that I wouldn't do it.
Time ago I bought a set of Chroma filter cubes for Nikon at bargain price but the filters were glued and I needed to dismount them to adapt the filters to my Zeiss cubes. I asked Chroma -very kind people- and they explained me how to dismount them, just with careful pressure as the glue is like a kind of very soft rubber. I was reluctant to do it -the filters are very delicate- but it worked well, I could forward you the instructions if you want.
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Pitufo



Joined: 21 Jun 2015
Posts: 171
Location: United Kingdom

PostPosted: Fri Feb 09, 2018 12:59 pm    Post subject: Reply with quote

Thanks Pau.

I realised I hadn't been very clear with my question and was in the process of editing it to include the world "subject".

I have a mercury lamp available so will try that for UV excitation in the short term. UV LED's sound like a much better idea though.

I did look at some plant cells today with my halogen with no success. I have just had a look at the halogen output spectrum and it must be too long wavelength to stimulate autofl in chlorophyll (I couldn't find the exact nm range during a quick search).


Last edited by Pitufo on Fri Feb 09, 2018 1:22 pm; edited 1 time in total
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ChrisR
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Joined: 14 Mar 2009
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Location: Near London, UK

PostPosted: Fri Feb 09, 2018 1:05 pm    Post subject: Reply with quote

Lovely image, though ( I checked in PS) the two images are almost a perfect match, so I don't see stereo either. Crying or Very sad
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Pitufo



Joined: 21 Jun 2015
Posts: 171
Location: United Kingdom

PostPosted: Fri Feb 09, 2018 1:26 pm    Post subject: Reply with quote

Thanks Chris.

The offset I used for the stereo was very small +1 and -1.

I originally used +2 and -2 and it came out looking horribly blurred.

Returning to filters: the reasons I was considering removing the emissions filter was to see if I could capture some red emission from chlorophyll and other sources.

I don't want to mess the cube up though - it is beautifully made.


Last edited by Pitufo on Fri Feb 09, 2018 1:32 pm; edited 1 time in total
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Pitufo



Joined: 21 Jun 2015
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PostPosted: Fri Feb 09, 2018 1:29 pm    Post subject: Reply with quote

Here's the +2/-2 cross-eyed stereo.

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Pitufo



Joined: 21 Jun 2015
Posts: 171
Location: United Kingdom

PostPosted: Fri Feb 09, 2018 2:20 pm    Post subject: Reply with quote

Amino acids NADH compounds, flavins, and chlorophylls appear to be the main fluorescing compounds in biological samples.

i.e.

- Leaf chlorophyll. The peak excitation wavelengths in the chlorophyll region are between 430 (emission 480nm?) and 490 nm (peak emission near 685 nm). Bacterial chlorophylls tend to emit further toward the infrared

- the fluorescent amino acids, tryptophan, tyrosine, and phenylalanine (excitation/emission maxima ~280/350 nm), which are constituents of many proteins;

- the reduced nicotinamide-adenine dinucleotides (NADH, NADPH) (excitation/emission maxima ~340/450 nm);

- flavin compounds (e.g., flavin adenine dinucleotide, flavoproteins, riboflavin) (excitation/emission maxima ~450/520 nm).

This data is taken from a pdf at the link below with some interesting excitation/emission 3D graphs of biological molecules.

It appears to be from some sort of US army project.

The link below is the direct PDF download of the research project (50 pages)

http://dtic.mil/cgi-bin/GetTRDoc?AD=ADA494347


Last edited by Pitufo on Fri Feb 09, 2018 2:40 pm; edited 7 times in total
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vasselle



Joined: 05 Jan 2014
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PostPosted: Fri Feb 09, 2018 2:26 pm    Post subject: Reply with quote

Very nice
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Pau
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Joined: 20 Jan 2010
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Location: Valencia, Spain

PostPosted: Fri Feb 09, 2018 3:35 pm    Post subject: Reply with quote

If you remove the emission filter you get:



At first look it seems very good and it is in 410nm cut but not so much at the long blue peak were you get up to 10% of the excitation light passing. Because fluorescence emission is much weaker than excitation this likely will produce a blue illuminated background obscuring fluorescence.
If dichroic beamsplitters where perfect emission filters will not be needed (and with lasers sometimes they are not used) but i fear this is not your case.
Substituting it with a longpass 500 or 510nm filter you will have good chlorophyll emission.

Sorry, the stereo still doesn't work for me

Thank you very much for the link.
Quote:
It appears to be from some sort of US army project

Quote:
Destroy this report when it is no longer needed

This sentence remembers me old spy films Laughing
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rjlittlefield
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PostPosted: Fri Feb 09, 2018 4:16 pm    Post subject: Reply with quote

Pitufo wrote:
I originally used +2 and -2 and it came out looking horribly blurred.

What are the physical dimensions of this stack? That is, how many microns wide, how many microns deep?

--Rik
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Jacek



Joined: 02 Oct 2011
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PostPosted: Sat Feb 10, 2018 3:48 am    Post subject: Reply with quote

Very nice
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Pitufo



Joined: 21 Jun 2015
Posts: 171
Location: United Kingdom

PostPosted: Sat Feb 10, 2018 5:34 am    Post subject: Reply with quote

Thank you.

I think the image field is 0.6mm but I'm not sure about the depth of the stack. I think it is very shallow which may explain why the stereos have not turned out well.
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Sumguy01



Joined: 28 Jan 2013
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Location: Ketchikan Alaska USA

PostPosted: Sat Feb 10, 2018 8:20 am    Post subject: Reply with quote

Very nice.
Thanks for sharing.
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rjlittlefield
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PostPosted: Sat Feb 10, 2018 9:37 am    Post subject: Reply with quote

Pitufo wrote:
I think the image field is 0.6mm but I'm not sure about the depth of the stack. I think it is very shallow which may explain why the stereos have not turned out well.

I agree, very shallow.

Running the numbers...

Assuming the Fluotar is 20X NA 0.45, then the quarter-lambda DOF is 0.0027 mm, so 25 frames would be around 0.068 mm, giving a depth/width aspect ratio of only 0.11.

Using the standard formulas , +-1% shift corresponds to stereo separation of 10 degrees. +-2 would be 20 degrees, too much to look good even if the images did not break up.

--Rik
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