Flattened Spirogyra

[75RR]

63x/1.4, DIC, 6 image stack in Photoshop

[zzffnn]

Very nice.
Did you have to use 0.12/0.13 mm cover (selected from #1 cover box)?

[75RR]

Thanks zzffnn,

coverslip box says 0.13 - 0.17mm
Zeiss manual says 0.17mm

[KurtM]

I'm lost. Is the weed flattened because it's being squished, or due to effects of image stacking? And what do the cover slips have to do with it (yes I know about objective lens compensating factor)??

Very cool image - one of these days maybe I'll shoot something that's still alive...

[zzffnn]

Maybe if 0.13 mm cover is used, the subject won't be flattened. IIRC, for oil objective, water mount is considered as part of the cover.

[mrsonchus]

Maybe if 0.13 mm cover is used, the subject won't be flattened. IIRC, for oil objective, water mount is considered as part of the cover.

Not quite sure what you mean - you've lost me a bit there old chap.

[mrsonchus]

Fantastic imaging, the details are astounding and beautiful - well done!

[75RR]

Thanks KurtM and mrsonchus

Think I flattened it when I attempted to photograph the Spirogyra zygotes

Maybe if 0.13 mm cover is used, the subject won't be flattened.

Don't have any, well not a box that says just that, though I suppose I could measure the ones I have and separate the thinner ones since I do have a micrometer.

Thought working distance was to the top of the cover slip.

[mrsonchus]

75RR - I personally wouldn't give cover-slip thickness another thought - it just isn't significant enough to worry about IMHO and experience - yes I use both for high-magnification x100 oil work, but I've never thought -'wow, that's a difference' in all honesty.

What I do however find is that I can get a far smaller amount and therefore a thinner layer of mountant to flow under the 0.13 cover-slips without problems, I use 19mm square and round a lot.
I've no idea why this is - it could simply be the finish of the glass with the 0.13s and not their thickness - but they do spread mountant beautifully.

[zzffnn]

I cannot remember exactly. But in the research world, scientists usually use 0.12-0.13 mm cover for 100x high NA objective.

Yes, I remember WD is calculated from top of cover.

Edit: Sorry, I think I got the cover thickness part (cited by John B) wrong. My poor memory confused it with water immersion objective viewing through balsam.

[zzffnn]

I think it is rather what an oil objective expects to see through vs. reality. I should not have called that cover thickness.

Expected (ideal) optical situation is: WD (air) + 0.17 mm cover + subject. Nothing else in between (subject touching cover slip), unless objective says no cover, of course.

Reality is:

In 75RR's case:
WD + cover + around 0.03 mm of water + subject

So if cover is 0.17 mm, you go over the ideal by 0.03 mm of water, which is optically equivalent to about 0.025 mm of oil. Assuming 75RR squeezes his cover as hard as I do (yes, I have measured my water mount thickness, after a very firm squeeze, with microscope fine focus travel - I got 0.03 mm). There is also spherical aberration caused by RI difference.

In John B's case, assuming mount's RI is close to oil/glass. You go over the ideal by whatever your mountant thickness is, if you use 0.17mm cover. Again, since John B has mostly stained samples mounted in high RI mountant, optical degradation will be harder to see.

So in both cases, 0.13 mm cover will help, as that leaves about 0.04 mm of mountant error-buffering space to be filled by mountant. After such a filling, the optical condition is then closer to the ideal (closer than when 0.17 mm cover is used).

Does that make sense now?

[mrsonchus]

That makes sense to me - thanks for that, it's very helpful. The 0.13s are also for what reason I don't know, very nice to handle and use. Very interesting reply. thanks.

[75RR]

Assuming 75RR squeezes his cover as hard as I do (yes, I have measured my water mount thickness, after a very firm squeeze, with microscope fine focus travel - I got 0.03 mm).

I don't squeeze! Well my objective does sometimes by accident!
I take it this is to both reduce subject to cover-slip distance and to make sure there is sufficient working distance (WD)?
How hard? How many times does that squish the subject? Risk of the equivalent of a road pizza ?

[gekko]

Beautiful image. Great detail and color. 755RR you are dong wonderful work with your DIC.
Squeezing cover glass: many years ago there used to be a mechanical device for doing this (I think it was called compressorium or something along those lines). I hope apochronaut sees this and perhaps give us more illumination on that.

[zzffnn]

Squeezing cover glass:

Yes, this is to reduce subject to cover-slip distance. I should call it a gentle tap (not "squeeze"!), as squeezing does more harm than benefit. Just once. Beacuase risk of crashing or removing protists are very high.

I found, through experiments, that it should be done as gentle as possible. Otherwise (when it is really a "squeeze" rather than "gentle tap"), some thick ciliates (many subjects filling 40x objective view field), such as Parameciums and Frontonias would be crashed. Also smaller protists may go away with water. And there is risk of evaporation and further compression, since you leave minimal amount of water under cover (once you squeeze, you need to scan and record as quickly as possible to avoid drying, even with LEDs).

A gentle tap may leave more than 0.03 mm thick of water under cover slip. I have not measured exactly how thick (40-50 microns, I would guess). We want intact and enough protists under cover slip first, so I guess optical condition comes second.

Rod probably has the best hands for gently compressing cover slips, using his coated slides. I remember he mentioned that he practiced such compression many times, some failed, but may times he managed to pin down protists for perfect camera pose. Exactly how to do it - please ask Rod - you can see that he has captured perfect poses more consistently than anyone else here. Maybe one can look at the subject, then slowly and gentle tap (compress) the cover while observing?

Those 30 micron hydrophobic coated slides will help too, as they make sure that you have at least 30 microns (if not more) of water under the cover (if you press tooooo hard, you may end up with even less than 30 microns, when coated slides are not used).

[75RR]

Those 30 micron hydrophobic coated slides will help too, as they make sure that you have at least 30 microns (if not more) of water under the cover (if you press tooooo hard, you may end up with even less than 30 microns, when coated slides are not used).

Good point

[KurtM]

Squeezing cover glass: many years ago there used to be a mechanical device for doing this (I think it was called compressorium or something along those lines). I hope apochronaut sees this and perhaps give us more illumination on that.

I'm certainly no apochronaut, but until he gets back here's this, from Ward & Whipple 1959 edition. Slide compressors are just another one of those tools that are seldom seen although one might think would be much more popular. Like rotating stages and oblique condensers, to name a couple.

[zzffnn]

^ That looks even better than Rod's hands (or Dalai Lama's secret spell) I want one! Along with a NA 1.4 oblique condenser!!

[75RR]

There is a modern version called the Taylor Microcompressor Mark II

http://www.microscopy-uk.org.uk/mag/ind ... ngly5.html

As to a Working Distance of 0.09mm, I think I basically just have to do a bit of prep work making sure the cover-slip is low enough.

[zzffnn]

I could not find the exact purchase link (price) for that Taylor Microcompressor though? http://www.norlandprod.com does not seem to have it anymore.

[charlie g]

Beautiful DIC image, 75RR, to my eyes. Tell me...with your 63X objective...does it have a correction collar adjustment ring?

I'm curious as to the engraved(or painted) spec on the objective barrel. Does it have the spec: "0.17" on the barrel?

I'm of understanding that: WD/working distance is the space between surface of coverslip and objective lens...not the distance between objective lens face and object on slide.

A lot can be sorted out by the exact specs engraved on objective lens barrel. Is this 63X objective with the spec:"DIC"?

Unlike balsam mounted slides...aqueous wetmount slides permit fantastic range of water film thickness under the coverslip,so with a high power and tight working distance objective...you start with a sensible water colum depth under the cover slip...but very rapidly this water film reduces...there is a sweet spot film thickness which permits 100X oil objective use on live organisms with no crush of the organism.

At 63X DIC objective..I ask if it has a correction collar as I wonder if this correction collar setting needs be set to range of your coverslips range of thickness? Before resorting to 'speced cover slips'...what spec is on the barrel of the 63X DIC objective you are useing for these great images, 75RR? Ahh..I'm thinking about DIC stand now! charlie guevara

[75RR]

Thanks Charlie g,

full text on the objective reads:

Planapo 63/1.4 Oel 160/-

No correction collar

This is a quote from this thread: http://www.photomacrography.net/forum/v ... hp?t=22425

The correct cover glass thickness should be No 1.5 (0.16-0.19 mm), not No 1 (0.13-0.16 mm). 0.17 mm is the reference cover glass thickness for all major manufacturers. That's why they also sell No 1.5H cover glasses (0.17 +/- 0.005 mm; actually for high NA immersion objectives; see below).
The Zeiss Planapo 63/1.4 Oil is marked "160/-". The "-" does not mean "tolerant to variation of coverglass thickness from 0.17" but "tolerant to variation of coverglass thickness from 0.17 taking into account NA and magnification".

Notice that there is a typo in the chart, the details on the 40x corr are those of the 100x

[gekko]

Kurt, my apologies! I should've said apochronaut or KurtM . Thank you very much for the information. And thanks also to 75RR for the additional information. No one seems to be making something like that anymore, am I right?

[rnabholz]

My ears were burning....

When using the hydrophobic slides to pin larger copepods or water fleas, I do not press the coverslip down. In fact I believe the reason that I can pin then successfully is that the cover floats a bit due to the water being prevented from spreading by the coating, actually creating a "dome".

The support of the water seems to provide just enough room underneath to immobilize the target without juicing them.

For what it's worth....

Rod

[gekko]

My ears were burning....
When using the hydrophobic slides to pin larger copepods or water fleas, I do not press the coverslip down. In fact I believe the reason that I can pin then successfully is that the cover floats a bit due to the water being prevented from spreading by the coating, actually creating a "dome".
The support of the water seems to provide just enough room underneath to immobilize the target without juicing them.
For what it's worth....
Rod

My experience with those slides is the same; however, I do press on them to seal the two compartments, and that would crush larger critters like crustaceans, but allows one to observe smaller microorganisms for a long time without worrying about the slide drying. Unlike Rod, I am not competent enough to be able to take photos of critters under a floating cover glass (hence shaking critters). Flash may be useful here.

[zzffnn]

With those coated slides, you can also add different volume to create different thickness ("dome size") for different subjects. Or use filter paper to suck out small amount of water to control "dome" thickness.

[KurtM]

Kurt, my apologies! I should've said apochronaut or KurtM

Nay sir - my knowledge is not to be compared with Phil's knowledge. I just happened to have a handle on this one, having run across it not long ago while searching for something entirely different, and pausing to regard it with interest and curiosity. Then when I saw it mentioned here, I was like the first grader who knows the answer in class, instantly shot my hand as high in the air as I could stretch my arm while gasping "oooh oooh ooooh ooooh I know I know I know I know"...

Or in other words, I could answer because I got lucky. Phil can answer because he's smart.

[charlie g]

Thanks for the terrific chart and showcase of variety of their line of objectives...I love to window shop!

But 75RR..the chart is for a 63X objective with spec: "160/ 0.17" your objective etched spec: " 160/-" implies: 'do not use with a cover slip!!?? What am I missing. Charlie guevara

[gekko]

... your objective etched spec: " 160/-" implies: 'do not use with a cover slip!!?? What am I missing. Charlie guevara

I believe it means that the thickness of the cover glass is not critical, and the objective may be used without one. I believe an objective that should not be used with a cover glass would be marked 160/0 or NCG.

I also thank 75RR for the chart, as I used to think that the working distance is measured from the tip of the objective rather than from the front lens as defined in the chart.

[charlie g]

Once I'm home...I can peek at my B&L metallurgical objectives...but they might actually print out the entire directive:"use no coverslip.". Charlie guevara