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morel squash prep

 
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discomorphella



Joined: 06 Jan 2005
Posts: 63
Location: NW United States

PostPosted: Sat May 27, 2006 10:46 am    Post subject: morel squash prep Reply with quote

Here's a simple recipe for preparing a morel; cut out a tiny piece of the mushroom, place it in a drop of PBS or water and squash the heck out of it with a coverglass (carefully). Although it doesn't have the uniformity of a paraffin or methacrylate section, and the microanatomy is a bit, well, distorted, its a fast way to see what you're eating. Note the immature spores in the ascus in the upper middle portion of the picture. One oblique and one phase contrast shot so you can see the different structures brought out by the 2 illumination conditions. Optical conditions:
12 V Halogen (Koehler)
Pl Apo 40X / 0.75
Periplan GW HP 10X relay
5 Mpxl DX4350 Kodak camera

--David



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Mike



Joined: 12 Dec 2004
Posts: 217
Location: Northeast Ohio, USA

PostPosted: Sun May 28, 2006 2:46 am    Post subject: Reply with quote

Hello David,

Two very nice shots! That PA 40X is working very well for you, indeed.

How are you accomplishing the oblique? I assume these were done on the Orthoplan. Howerver you did it, it really looks very DIC'ish and I'd like to give it a try.

All the best,
Mike

p.s. - thanks for the fluorescence cube info.
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discomorphella



Joined: 06 Jan 2005
Posts: 63
Location: NW United States

PostPosted: Sun May 28, 2006 8:18 am    Post subject: Reply with quote

Thanks Mike--
The oblique shot was illuminated with a semicircular disk (black plastic sheet) in one of the slots in the 402a condenser turret on the orthoplan. It was ~1/3 of the radius. As Ron Neumeyer has pointed out you can get a very similar pattern by partially offsetting the BF aperture as well, but its just takes a bit more effort to get a reproducible effect. I suppose you could make a paper scale to attach to the 402 and calibrate it using the Bertrand lens on your variotube. Hmmm. In fact I think I might do that too...it will be easier than opening that little hatch on the 402a.
It also helps to have an objective with a correction collar or keep the specimen pretty thin, none of these off-axis techniques really tolerate much specimen thickness. The mushroom prep was very squashed so the thickness wasn't a problem, but I would typically use an objective with a correction collar for oblique with most wet mounts.

Regards,

David
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Charles Krebs



Joined: 13 Jul 2004
Posts: 1200
Location: Issaquah, WA USA

PostPosted: Sun May 28, 2006 12:37 pm    Post subject: Reply with quote

David... excellent in both cases! The phase shots you post look so "clean" (for lack of a better word). One part of that is probably your good sample preparation. Flatter definitely is better in phase.
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micron



Joined: 29 May 2004
Posts: 294
Location: Delta, BC - Canada

PostPosted: Sun May 28, 2006 3:49 pm    Post subject: Reply with quote

Hi David,

Very nice examples of oblique and phase contrast. I will have to try making a stop for my condenser; with my setup offsetting the aperture diaphragm does not always produce the best oblique effect.
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discomorphella



Joined: 06 Jan 2005
Posts: 63
Location: NW United States

PostPosted: Mon May 29, 2006 12:21 am    Post subject: Reply with quote

Thanks Ron and Charlie and Mike for your kind replies. One thing to keep in mind for oblique is that the background illumination "cleanliness" is an important factor in the final image. I typically make sure I get a good blank image before I start a run of oblique shots and then use imageJ to normalize out the background. Same thing for phase. I think Ron's trick of offsetting the brightfield aperture works for most specimens, but if I get the time I'd really like to figure out what the optimal family of aperture shapes is for a given objective NA and sample.

David
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Ken Ramos
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Joined: 04 May 2004
Posts: 4809
Location: Western North Carolina

PostPosted: Mon May 29, 2006 4:19 pm    Post subject: Reply with quote

Now these are some striking images. Why can't I get results like this? Laughing Good work David and great shots! Very Happy
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discomorphella



Joined: 06 Jan 2005
Posts: 63
Location: NW United States

PostPosted: Mon May 29, 2006 11:38 pm    Post subject: Reply with quote

Thanks Ken--
And I seem to recall you do get images like these Laughing . I just wish I had a higher success rate, I have to collect gigabytes of "nonstriking" images... to obtain these few decent ones..

--David
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