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Proper darkfield microscopy?

 
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Thomas Ashcraft



Joined: 31 May 2006
Posts: 25

PostPosted: Fri Jun 09, 2006 2:06 pm    Post subject: Proper darkfield microscopy? Reply with quote

First, thank you all for your patience in answering my questions. These are questions that I haven't found the answers to on the web after much digging and googling.

So then, what are the best objectives for darkfield imaging? Brightfield objectives stopped down? Or phase contrast objectives stopped down?

Is a Zernike condenser better for darkfield imaging than a standard achromatic substage condenser 1.25 NA with a darkfield stop?

When I look at videos of microbes ( for example at Molecular Expressions archives - see url below as example) I find I am more attracted to a rich black darkfield background rather than the grays of phase contrast and plan to head in this direction in my work.

http://146.201.224.61/moviegallery/pondscum/protozoa/dileptus/index.html

So...Thanks again! - Thomas
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Thomas Ashcraft



Joined: 31 May 2006
Posts: 25

PostPosted: Fri Jun 09, 2006 3:21 pm    Post subject: Reply with quote

Answering my own questions : I just discovered some excellent darkfield setup tutorials and primers at:

http://micro.magnet.fsu.edu/primer/techniques/darkfield.html

http://micro.magnet.fsu.edu/primer/techniques/darkfieldsetup.html

Smile
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psneeley



Joined: 11 Dec 2004
Posts: 23
Location: Utah

PostPosted: Fri Jun 09, 2006 6:30 pm    Post subject: Reply with quote

The very best DF is gotten by using an oil immersion DF condenser oiled to the bottom of the slide. You'll need this to reach N.A. 0.85 or so. After that, you probably need very special DF condensers or must have an iris diaphragm (or DF cone inserted in the objective) to limit the N.A.

A oil immersion, DF condenser will provide a very, ultra-black field at 40x, and 20x, and often 10x (if it is a Wide Field condenser too)

Any objective will do, but DF is very demanding on optics and tends to show any error in correction that would get lost in BF.

It -is- beautiful though, and even a DF stop in the condenser will yield good results for the 10x and 20x objectives.

DF is not everything-- If you are wanting to look at pond water for tiny beasties, Phase is very, very useful.

I spend most of my scope time gazing at pond water using phase. I admit, though, that the most magical moments I've had have been using oil immersion DF looking between the beasties into the world of spirochetes and bacteria -- sometimes, if things are just right, you can see a lot there.

I've said this before, in another thread here perhaps, but I find some amoeba to be almost transparent in DF. Phase is the best method for these.

My experience is admittedly limited, but the above is what I've found to be true.

FWIW,

Steve
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Thomas Ashcraft



Joined: 31 May 2006
Posts: 25

PostPosted: Sat Jun 10, 2006 6:25 am    Post subject: Reply with quote

Steve wrote:

"DF is not everything-- If you are wanting to look at pond water for tiny beasties, Phase is very, very useful."


Steve and all,

Can you do decent if not optimum darkfield then going through phase contrast objectives with a darkfield stop in the condenser (and Koehler illumination)?

I ask because I am looking to buy a microscope that is a phase system with 10x -20x-40x phase contrast with darkfield stops at those magnifications. But I am wondering if darkfield works better through BF objectives rather than phase contrast objectives?

I haven't been able to figure this out reading the online tutorials as yet.

Thomas
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psneeley



Joined: 11 Dec 2004
Posts: 23
Location: Utah

PostPosted: Sat Jun 10, 2006 9:48 am    Post subject: Reply with quote

A Phase Condenser is a good BF condenser too. You can get DF from using the 40x annulus on the 10x objective (really, COL, but DF just the same). The 20x may give you good DF with 40x or 100x annulus -- you'll just have to try. If the phase condenser has a filter holder, you certainly can insert a DF stop there and it should work. Some phase condensers have a DF stop right in the slot an annulus might be -- usually, the annulus slots being Open (BF), DF, 10x, 20x, 40x, 100x. By rotating an annuli off of center -- to almost the edge -- you can get good obique effects too, at least for the 10x and 20x objectives.

BUT, a true, oil immersion, DF condenser will furnish the blackest and finest DF.

Work with what you have -- a good phase condenser (pancake, turret style would be the best) will be very versatile.

FWIW,

Steve
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Thomas Ashcraft



Joined: 31 May 2006
Posts: 25

PostPosted: Sun Jun 11, 2006 8:41 am    Post subject: Reply with quote

Steve wrote: " Work with what you have -- a good phase condenser (pancake, turret style would be the best) will be very versatile."

Steve,

Thanks again for your input. It is very helpful.

Right now I am working with an old ( 1930s maybe ) Spencer Buffalo microscope that has seen its day but is still worthy of some good science. I got it used from a University and I'm sure over the decades the 95x objective crunched into many a cover glass! But it still produces a useable image. ( Tip of the hat to the old Spencer craftsmen.)

I plan to get a new microscope soon but needless to say it is a costly endeavor. So I am trying to get it as correct as possible from the onset. Though I am seeing that over time it will be necessary to acquire various other objectives and specialty condensers.

Thomas
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psneeley



Joined: 11 Dec 2004
Posts: 23
Location: Utah

PostPosted: Sun Jun 11, 2006 10:02 am    Post subject: Reply with quote

You might consider an AO Spencer Series 10 (infinity corrected), or an AO series 4 (160mm) from ebay, or from another member of this forum or the Yahoo! Microscope forum. In fact, you might consider getting both. Rationale:

1. They are strongly made
2. Objectives are plentiful on ebay and elsewhere
3. Condenser are plentiful -- the DF Condenser made for the Series 10 will fit the Series 4 too -- and they go cheap on eBay.
4. Phase condensers are relatively plentiful for these models -- you just have to have patience and watch ebay.
5. They are cheap -- outrageously so.
6. True, the factory that made them has gone out of business, but when it was running, it produced many scopes that are just now coming out of use in may labs, hospitals, and universities -- this makes for cheap prices
7. There are lots of tech people who can repair them for you.

But, if you want an excellent alternative, Nightingale microscopes has Leitz SM's Phase scopes for $1400. Refurbed to new. These would get you immediately into the phase world, and DF condensers for Leitz models are very plentiful too. As are objectives, etc.
See:

http://www.microscopesfromnightingale.com/cgi-bin/store/agora.cgi?cart_id=9524940.59067*hn3j-6&p_id=arc_leitz_phase_contrast&xm=on&ppinc=display

FWIW,

Steve
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Thomas Ashcraft



Joined: 31 May 2006
Posts: 25

PostPosted: Tue Jun 13, 2006 5:50 pm    Post subject: Reply with quote

[Steve wrote: "You might consider an AO Spencer Series 10 (infinity corrected), or an AO series 4 .......But, if you want an excellent alternative, Nightingale microscopes has Leitz SM's Phase scopes for $1400. Refurbed to new. " )


Steve,

Thank you for your considerable advice. It is highly appreciated. Foraging through the various microscope possibilities is an arduous process and I value the experience of those like yourself who have gone before me and generously report their findings. I know there are well worn paths through Ebay, Nightingale, Martin, and new scope dealers.

I am bleary eyed from studying all the messages in the archived Microscope yahoo group but am getting informed in the process. Kind of fun actually.

Thanks! Good observing to you.

Thomas
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