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Best DIC set-up for cell details with wet mounts?
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descall



Joined: 01 Oct 2011
Posts: 167
Location: United Kingdom

PostPosted: Tue Nov 14, 2017 9:22 am    Post subject: Best DIC set-up for cell details with wet mounts? Reply with quote

I’m ruminating over what DIC set-up I should invest in for photographing the cell structure of living moss leaves, wet mounted, wanting to capture as much detail as possible. The below shows a typical subject, a photo I captured a few years ago with a DIC system I once owned (Olympus BX51, UPlanFL N 60x/0.9 dry, Canon EOS T3i direct projection). I want to do better than this. I’m thinking of returning to Olympus, simply because I have experience with the system, though I’m happy to change my mind. So, I guess, I have three main questions:
1. Which objective would be best for my requirement, bearing in mind it will be a DIC system?
2. Would I resolve much more detail with 100x vs 60x (i.e. could I stick with the best 60x only)?
3. Which camera set-up would be best (direct projection onto APSC or FF via a relay lens)?

Many thanks for any help :-)

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Pau
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Joined: 20 Jan 2010
Posts: 4008
Location: Valencia, Spain

PostPosted: Tue Nov 14, 2017 9:55 am    Post subject: Reply with quote

Resolution doesn't depend of magnification but of NA. You can't get more from a 100/1.4 than from a 60/1.4, again the brand is not relevant (assuming Plan Apos from any of the big four)

Some systems (Oly and Nikon for sure) make low contrast DIC prisms aimed to a gain a bit resolution advantage over high contrast ones.

The condenser also matters, use an oil immersion 1.4 one if available

If you use deep blue light resolution also can improve (and you lose colors)

Intact leaves with water immersion is not the best recipe to reach resolution limits although it provides very nice images. A well prepared and stained thin section could resolve a bit more.

But for even higher resolution you'd need to abandon DIC, go to fluorescence confocal, superresolution systems or TEM

Any modern mirrorless or DSLR has enough megapixels to easily outresolve the objectives at this magnification range. Your former system seems excellent.
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descall



Joined: 01 Oct 2011
Posts: 167
Location: United Kingdom

PostPosted: Tue Nov 14, 2017 12:52 pm    Post subject: Reply with quote

Thank you Pau, that's very useful indeed :-)
Below is one system, based around the BX53, that I am considering (haven't priced it yet...). Would you make any changes to this? I would use any old 10x objective as a finder lens for the moment, buying further objectives in the future. The 60x oil provides NA 1.35 and the oil top lens on the condenser NA 1.4. I wonder what is the difference between the U-DICT and U-DICTS sliders, in terms of image quality? No DIC prisms match the UPlanSApo 60x oil with the U-DICT slider, hence my provisional choice of the U-DICTS slider. I would intend to mount a Canon 700D on the top, direct projection.

Many thanks for any further comments.

WHN10X Eyepieces
U-TR30-2 Trinocular tube
U-CA Magnification changer
BX3-ARM Standard arm
BX53F2 BX53 Frame
U-D6RE Sextuple revolving nosepiece for DIC/simple POL
U-DICTS Shift DIC slider
UPlanSApo 60X/1.35 oil objective
U-SVRB-4 Mechanical stages with right-hand control
U-HLD-4 Specimen holder
U-UCD8-2 8-position universal condenser
U-TLO Oil top lens
U-ODIC60 DIC element
U-LHLEDC100 High power LED lamp housing
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Charles Krebs



Joined: 01 Aug 2006
Posts: 5762
Location: Issaquah, WA USA

PostPosted: Tue Nov 14, 2017 5:58 pm    Post subject: Reply with quote

I would very seriously consider the UPLSAPO 60XW (see http://nimax-img.de/Produktdownloads/49976_uplsapo_60xw_en.pdf )

With wet mounted subjects (as you mentioned above), one of the biggest killers of image quality when using high NA oil objectives is a water filled gap between the subject and the cover glass. Ideally you want all of the subject in contact with the cover glass, but often (usually?) with anything other than a totally flat subject this is not possible. The spherical aberration can get pretty severe. Water immersion objectives like this one help a great deal.

Good explanation and discussion here:
https://www.microscopyu.com/microscopy-basics/water-immersion-objectives
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descall



Joined: 01 Oct 2011
Posts: 167
Location: United Kingdom

PostPosted: Wed Nov 15, 2017 1:31 am    Post subject: Reply with quote

Brilliant help Charles, many thanks indeed Very Happy I shall switch plans to the UPlanSApo 60XW. The matching DIC prism is the same (U-ODIC60) but I now have a choice of sliders U-DICT and U-DICTS, the latter providing 'shift type' DIC. I wonder which is the best for my purpose?

Also, I assume an oil top lens on the condenser (NA 1.4) is still the best option, rather than dry (NA 0.9).

Thanks again for your valuable help :-)
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Pau
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Joined: 20 Jan 2010
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Location: Valencia, Spain

PostPosted: Wed Nov 15, 2017 2:21 am    Post subject: Reply with quote

Although theoretically (ideal conditions) a 1.2 objective could deliver less resolution than a 1.35 one the devil is in the details, I think Charles is right. I always rely in his experience and knowledge.

About the DIC prism AFAIK U-DICTS is about the same than U-DICT but can switch vertically the prism between two positions to be compatible with a wider range of objectives. U-DICT can hold its own analyzer U-ANT while U-DICTS needs and independent analyzer slider. There are also other objective DIC prisms:
- U-DICTHR High-resolution DIC slider for transmitted light
- U-DICTHC High-contrast DIC slider for transmitted light
Maybe for your requirements with thick specimens the U-DICTHR could be the most adequate if compatible with your chosen objectives.
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JohnyM



Joined: 24 Dec 2013
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PostPosted: Wed Nov 15, 2017 4:50 am    Post subject: Reply with quote

Or you can mount those in immersion oil and enjoy 1.4 NA lens instead of troublesome correction collar WI.
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zzffnn



Joined: 22 May 2014
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Location: Texas USA

PostPosted: Wed Nov 15, 2017 5:59 am    Post subject: Reply with quote

If you don't need to keep those moss leaves or thin sections after viewing/photographing under microscope, then you may indeed mount them in immersion oil to get NA 1.4. Best to wipe them dry and rinse at least twice in oil to replace all surface water (with oil).

If surface water cannot be reliably removed, then water immersion objective may work better.
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Pau
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Joined: 20 Jan 2010
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Location: Valencia, Spain

PostPosted: Wed Nov 15, 2017 6:19 am    Post subject: Reply with quote

zzffnn wrote:
If you don't need to keep those moss leaves or thin sections after viewing/photographing under microscope, then you may indeed mount them in immersion oil to get NA 1.4. Best to wipe them dry and rinse at least twice in oil to replace all surface water (with oil).


Did you actually try it with leaves?
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JohnyM



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PostPosted: Wed Nov 15, 2017 6:49 am    Post subject: Reply with quote

Im mounting living cambium stripes in immersion oil with no problems. I was using 60x 1.4 now im using 40 1.3 and couldnt be happier. Also if im using water mounted specimen 1.3 lens is giving greater penetration depth.
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sushidelic



Joined: 10 Jun 2016
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PostPosted: Wed Nov 15, 2017 7:16 am    Post subject: Reply with quote

U-DICTS is not the same as U-DICT, you are not able to turn it into colorful interference colors as you can do with the U-DICT.

See here on page 15:
http://cn.olympus.com/upload/accessory/20114/201147144618944709.pdf

I will have a look if I find an appropriate slide to test (maybe some thin section), and would love to do a comparison between U-DICT, U-DICTS, U-DICTHR and U-DICTHC. Time is the problem at the moment, but I'll try.

Here's a another very basic PDF for the different Oly - DICs
http://www.samkyung21.com/file/notice/pdsdown.asp?pseq=467&filenm=DIC%2Epdf&filegu=&tbl=board49

Best regrds,
Michael
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Pau
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Joined: 20 Jan 2010
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Location: Valencia, Spain

PostPosted: Wed Nov 15, 2017 10:26 am    Post subject: Reply with quote

Michael, thanks for the info, I only have the U-DICT (and I use it in a hybrid setup), and I only knew the Nomarski DIC slider Lineup leaflet where the difference between U-DICT and U-DICTS is not explained (you could get my same conclusion reading it alone)

In personal communication Carlos told me that the lever at the U-DICTS just lowers the prism about 6mm

Quote:
I will have a look if I find an appropriate slide to test (maybe some thin section), and would love to do a comparison between U-DICT, U-DICTS, U-DICTHR and U-DICTHC. Time is the problem at the moment, but I'll try.

It would very nice to see that comparison. The images at Olympus pages have too low resolution
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Pau
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PostPosted: Wed Nov 15, 2017 10:35 am    Post subject: Reply with quote

Thanks Fan and Johny for your oil immersion ideas, a thing I will try.
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Beatsy



Joined: 05 Jul 2013
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Location: Malvern, UK

PostPosted: Wed Nov 15, 2017 2:59 pm    Post subject: Reply with quote

Pau wrote:
Resolution doesn't depend of magnification but of NA. You can't get more from a 100/1.4 than from a 60/1.4


This prompted a thought (can you smell smoke Smile ). While I totally agree with the statement above, I wonder if it still holds true with DIC? As I understand it, you lose some resolution along the direction 90 degrees to the shear axis with DIC. I proved that to myself with radially mounted test diatoms where the ones with punctae parallel to the shear axis showed separation - those at 90 degrees to it didn't. I can't find the picture right now. Anyway - this made me wonder if using higher (empty?) magnification at a given N.A. might dodge some of this "resolution robbing" compared to a lower mag. Probably not - but...
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JohnyM



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PostPosted: Wed Nov 15, 2017 3:52 pm    Post subject: Reply with quote

Nope, shearing amount is fixed to slider.
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