First attempt with Water Immersion

[hkv]

I finally received my new 60X Water Immersion objective today. Decided to give it a try and took an old sample where Algae is thriving.



The resolution is not as good as 60X oil (NA 1.35), but this one has NA 1.20. The advantages of not having to mess with oil is well worth the loss in resolution I think. At least in the majority of the situations.

First image! I have no clue what type of algae this is, but it is a colony held together by mucilage and it had fast moving flagella.
Stack of 12. Decent DIC effect with a mismatched prism (Still does not have the correct 60X prism)

[gekko]

To my eyes, this looks a fantastic image, with wonderful DIC effect, color, and detail.

[zzffnn]

Very nice DIC image and congratulations!

[kit1980]

Very nice!

[Johann]

Very nice - will take that any day over the hassle of oil

[charlie g]

Fantastic image capture of a colony of Gonium...(G. pectorale ?), thanks hkv. Did you adjust of 'twiddle a bit' with the correction collar on this WI objective? Did you sense image quality responses to adjusting this correction collar? I always am unsure with my correction collar objectives...just where to start (so I start at 'middle' of the collars range!).

Your stand makes me drool..great instrument...thanks for these visits to your bench.


I always wonder at the variety of specs on our objective barrels..please coment what: UPlan SApo means? I know it's an infinity-plan-apo objective with correction colar. What is the "U" and "S" terminology? Thanks for your shared microscopy,hkv. charlie guevara

[hkv]

Fantastic image capture of a colony of Gonium...(G. pectorale ?), thanks hkv. Did you adjust of 'twiddle a bit' with the correction collar on this WI objective? Did you sense image quality responses to adjusting this correction collar? I always am unsure with my correction collar objectives...just where to start (so I start at 'middle' of the collars range!).

Your stand makes me drool..great instrument...thanks for these visits to your bench.


I always wonder at the variety of specs on our objective barrels..please coment what: UPlan SApo means? I know it's an infinity-plan-apo objective with correction colar. What is the "U" and "S" terminology? Thanks for your shared microscopy,hkv. charlie guevara

Thanks for looking! And thank you for the ID! I wonder how you experts manage to find the right ID all the time? I have no clue where to start... I need a reverse image google search engine, but the ones that exist are completely useless. I will make a separate post on this topic someday.

Well, in this case I used a #1 cover slip. I tweaked down the collar to 0.13, but I got a better imager with a slightly higher number. Not all the way up to 0.17 though. It is always a hassle because when you change the collar, you need to refocus and then try to remember if it got better or worse since last setting. BUT, I can definitely see a big difference when I adjust it.

UplanSAPO is Olympus most versatile APO series. "SAPO" stands for "Super APO" meaning meeting their highest standards. The "U" stands for "Universal". It means they are good for DIC, POL, Bright Field, Fluoresence, Dark Field (lower NA). Chromatic aberration is close to zero, at least I have never noticed any. I see more purple fringes with the PlanFL (Fluorite) series, but very easy to remove in Photoshop. In all, the UplanSAPOs are very good objectives and I enjoy them every time I use them! The 60X water immersion is fantastic because it has a whopping 0.28 mm working distance which allows rather thick water films and specimens. Almost double the W.D compared to the oil version.

[zzffnn]

hkv's scope photo shows the beauty of a water immersion objective - you can put a dry NA 0.95 objective right next to it and switch between them within a few seconds. Yes, seconds, not minutes! Simply soak away immersion water with a cotton swap.

Try the same switching between an oil objective to a dry NA 0.95 objective. I did.

The clean-up may take 2 minutes or more (your protist may already swim away), even if you clean up the oil reasonably well and manage not to compress most of your water sample away. The former was not too difficult, when I used another blank/clean cover slip to scrape away the immersion oil. But the consequential sample compression (your protist being crashed or flooded out) is hard to avoid, even if you try to be careful. Not to mention residual oil (which is nearly impossible to completely remove) will leave a slight fog when you view the slide under your NA 0.95 dry apo.

[hkv]

hkv's scope photo shows the beauty of a water immersion objective - you can put a dry NA 0.95 objective right next to it and switch between them within a few seconds. Yes, seconds, not minutes! Simply soak away immersion water with a cotton swap.

Try the same switching between an oil objective to a dry NA 0.95 objective. I did.

The clean-up may take 2 minutes or more (your protist may already swim away), even if you clean up the oil reasonably well and manage not to compress most of your water sample away. The former was not too difficult, when I used another blank/clean cover slip to scrape away the immersion oil. But the consequential sample compression (your protist being crashed or flooded out) is hard to avoid, even if you try to be careful. Not to mention residual oil (which is nearly impossible to completely remove) will leave a slight fog when you view the slide under your NA 0.95 dry apo.

Yes, the upside is tremendous. Fully agree. I see in the footer that you are selling yours? Missing the oil mess?

[hkv]

Fantastic image capture of a colony of Gonium...(G. pectorale ?), thanks hkv.

Could it be Gonium Sociale? Looks just like when looking at other pictures.

[hkv]

Charlie, any idea what this is?

[zzffnn]

hkv,

I currently have 5 LOMO water objectives (30/0.9, 40/0.75, 65/1.1 and two 70/1.23). So one of my 70/1.23 apo is now a surplus and functional duplicate. That is why I am selling it.

Another way to avoid oil mess is not to clean it up at all, by using low power oil immersion objectives. I have another nose for oil objectives, 10x, 30x, 60x and 90x.

[Lyra23]

It's look like photoshop painting. Your DIC image is very nice!

[charlie g]

Fantastic image capture of a colony of Gonium...(G. pectorale ?), thanks hkv.

Could it be Gonium Sociale? Looks just like when looking at other pictures.

As always, hkv..we (myself especially!) enjoy and learn from your shared microscopy..again thanks!

Gonium sociale is agreed to be a four (4) celled species..my most recent cluttered library content suggests there are perhaps two known four celled (4) subtypes of Gonium sociale.

There is terrific pleasure when I make time to look up these neighbors we all share our world with...inspired by microscopy posts here in forum...again thanks,hkv. It's great that my 1904 text(G.S.West), my 1950 text (G.M.Smith), and my 1954 text( G.W.Prescott) all agree, all build on the habits of this common species (our dear neighbors!) of mudd puddles and freshwaters of ditches, etc. charlie guevara

[hkv]

Charlie, you are right. I studied some of the old books I found online and you are absolutely right. Gonium Sociale has 4 cells only. I stand corrected! Gonium Pectorale it is.

[billben74]

Top class. Interesting to see your water immersion.
I've often wondered why they aren't more common than oil lenses.
It may simply be tradition.
I get the resolution thing but the waters seem more practical
for most purposes.
Yours is working beautifully.

[charlie g]

Top class. Interesting to see your water immersion.
I've often wondered why they aren't more common than oil lenses.
It may simply be tradition.
I get the resolution thing but the waters seem more practical
for most purposes.
Yours is working beautifully.

Hi billben74..at risk of highjack of hkv's post ..err..this wonderful WI objective is the joy of this thread..and WI objectives still carry heavy work duties in microscopy of live tissues..you can't have oil-immersion objectives in contact with live tissues.

Ahh but if only to go back to school at this time! charlie guevara

[charlie g]

Fantastic image capture of a colony of Gonium...(G. pectorale ?), thanks hkv. Did you adjust of 'twiddle a bit' with the correction collar on this WI objective? Did you sense image quality responses to adjusting this correction collar? I always am unsure with my correction collar objectives...just where to start (so I start at 'middle' of the collars range!).

Your stand makes me drool..great instrument...thanks for these visits to your bench.


I always wonder at the variety of specs on our objective barrels..please coment what: UPlan SApo means? I know it's an infinity-plan-apo objective with correction colar. What is the "U" and "S" terminology? Thanks for your shared microscopy,hkv. charlie guevara

Thanks for looking! And thank you for the ID! I wonder how you experts manage to find the right ID all the time? I have no clue where to start... I need a reverse image google search engine, but the ones that exist are completely useless. I will make a separate post on this topic someday.

Well, in this case I used a #1 cover slip. I tweaked down the collar to 0.13, but I got a better imager with a slightly higher number. Not all the way up to 0.17 though. It is always a hassle because when you change the collar, you need to refocus and then try to remember if it got better or worse since last setting. BUT, I can definitely see a big difference when I adjust it.


Hkv, 'please do tell'!? On my quintuple turrent of objectives..it's quite a trick to get fingers to adjust my correction collar on one 'fat objective barrel' ..amidst the forrest of the four other 'fat objective barrels'...do you, hkv ,have this chore as a challenge also? I ask as your kind post of this optimum workhorse stand displays a crowded forrest of 'fat objective barrels' surrounding you new 60X WI/correction collar objective. How do you twiddle and adjust that correction collar amidst your nosepiece of objectives..or does your workhorse stand have 'room to spare' between the objectives? I also ask your opinion on the microscopes which have...gulp!...sextuple turrent of objectives..how on earth do human fingers adjust correction collars with wetmount slide preps for these stands?!

Do you water-bridge the substage condenser to the slide bottom..or leave it air-spaced? Would it be practicle to try either oil-bridge , or water bridge, of the substage condenser to your wetmount slides..to see if there is notable enhancement of your images?

Is your premium apo objective demanding an adjustment for water colum thickness beneath the #1 coverslip you use..from wetmount slide to the next wetmount slide you observe..or do you (being familiar with the at times tight working distanc of your dry objectives)..do you try and maintain a 'sweet spot' minimal water film thickness in you wetmount slide observations.

Guess I,m asking how ofter , in wetmount slide observations, do you need to again readjust the correction collar objectives you enjoy for WI objectives..or 'high dry/high NA objectives'?

I drank up your kind DIC posts..as well as this current post, thanks,hkv..very sorry for the Stockholm tragedy..warmly, charlie guevara, finger lakes/US

[hkv]

Hkv, 'please do tell'!? On my quintuple turrent of objectives..it's quite a trick to get fingers to adjust my correction collar on one 'fat objective barrel' ..amidst the forrest of the four other 'fat objective barrels'...do you, hkv ,have this chore as a challenge also? I ask as your kind post of this optimum workhorse stand displays a crowded forrest of 'fat objective barrels' surrounding you new 60X WI/correction collar objective. How do you twiddle and adjust that correction collar amidst your nosepiece of objectives..or does your workhorse stand have 'room to spare' between the objectives? I also ask your opinion on the microscopes which have...gulp!...sextuple turrent of objectives..how on earth do human fingers adjust correction collars with wetmount slide preps for these stands?!

Well, I agree. Fingers are a bit chubby to get in between, but the diameter of the turret is quite large and I can get two fingers in between. Normally I use the finger tips of two fingers, one from each hand, to twist the ring. Works ok.

Do you water-bridge the substage condenser to the slide bottom..or leave it air-spaced? Would it be practicle to try either oil-bridge , or water bridge, of the substage condenser to your wetmount slides..to see if there is notable enhancement of your images?

No I did not. It was an air gap. I have an oil condenser also, but have not tried to apply a water bridge. I will do that and see if things improve. To use oil would be worthless, as then I am back at the oil mess. I have not yet figured out weather the NA 1.2 can be achieved through oil or water, but it would be impossible (I think) to reach NA 1.2 with an air gap between the condenser and glass.

Is your premium apo objective demanding an adjustment for water colum thickness beneath the #1 coverslip you use..from wetmount slide to the next wetmount slide you observe..or do you (being familiar with the at times tight working distanc of your dry objectives)..do you try and maintain a 'sweet spot' minimal water film thickness in you wetmount slide observations.

I always try to have as little water columns thickness as possible. Using the APO 40X and above is very sensitive to water thickness. The 60X WI, have a W.D of 0.28 mm is much more forgiving. S

Guess I,m asking how ofter , in wetmount slide observations, do you need to again readjust the correction collar objectives you enjoy for WI objectives..or 'high dry/high NA objectives'?

I drank up your kind DIC posts..as well as this current post, thanks,hkv..very sorry for the Stockholm tragedy..warmly, charlie guevara, finger lakes/US

I adjust it every time. Every wet mount is different and needs to be adjusted. Sometimes it is impossible to get good resolution or removal of halos and I just redo the mount with a new cover glass and less water.

The Stockholm attack was terrible. I live in Stockholm and pass that area more or less every day. Times are changing.

[RudiV]

Really great results hkv!

The water immersion objective is really working well, I think I need one...

Love your images, gives me something to aspire to!

Thanks for sharing and motivating,
Rudi

[hkv]

Really great results hkv!

The water immersion objective is really working well, I think I need one...

Love your images, gives me something to aspire to!

Thanks for sharing and motivating,
Rudi

Thanks for looking! Yes, I am really happy with the performance so far and I have only tried it once... Will be a new favourite.

[KurtM]

That first image is absolutely top drawer as far as I'm concerned. Those "fast moving flagella" (present in many algae species including the famous Volvox) are always a challenge object visually, more so in imaging, and you flippin' NAILED it buddy! And for a mismatched DIC prism, I see little to be vexed over. I see a lovely DIC effect all the same. I am in awe ... and envious ... of your equipment!

[hkv]

Thanks Kurt! The flagella was a pain to get right. In each fram of the stack a part of a flagella was in focus, so it took some "retouching" in Zerene to extract the focused ones into the final image. I did not use a flash, so it I needed to keep the exposure time a little higher than I wanted to at the same time keeping the ISO low. With the Canon 6D I can go to ISO 1000 without too much noise. At 1600 it starts to get really bad and a lot of smoothing is needed to get a decent result.