"OEM Nikon 20x 0.75 VC" cover slip thickness

Lou Jost · Post by **Lou Jost** » Sun Dec 30, 2018 7:07 am

Abednego, can you tell us more? The patent is unclear about whether the objective is designed to be focused on the front inner surface of the cell, or the rear inner surface, or at a compromise point near the center of the cell. Do you know the thickness of the cell wall and its depth?

zzffnn · Post by **zzffnn** » Sun Dec 30, 2018 11:31 am

Forum members who commented on this thread likely all know this, but I will add it for other casual visitors.

Even #1 cover slips made in China these days can be pretty good. I measured 10 AmScope #1 cover slips previously, with a micrometer, and found only one 0.15 or 0.14mm (the other 9 were all at 0.17mm). I also measure 10 #1.5 Zeiss cover slips and found all to be 0.17mm. Measure your cover slip to be sure.

Liquid column depth can be measured by microscope: focus onto top surface of microscope slide, note the fine focus location, move up fine focus into good focus on bottom of cover slip, note find focus location again and calculate that depth difference (which is the liquid column depth).

I measured a very firmly compressed layer of water between cover slip and slide before and got a liquid depth of about 30 microns.

One can easily change water to immersion oil and measure depth the same way. You would likely have more liquid column depth, if you use thicker liquid and compress it less.

Lou,

A patent or applicant would never want to specify anything to limit its utility, unless a patent examiner asks it to be done (to make it easy for people to use the patent later on). Maybe you can ask manufacturer or sale agent the exact technical specifications, if you cannot test it yourself?

I would guess the gene sequencer 's microscope objective is designed to focus onto the actual sample (chip?) surface. But that extra ~60 microns (?just guessing) of fluid depth may not matter that much at NA 0.75, if cover slip correction of the objective is designed to be 400 microns? NA 0.75 is right where one would start to really worry about cover slip correction though, so you may want to ask an optician for the exact numbers/math. Maybe Rik would be able to give you some numbers (he is very good at optical calculations, as you all know).

Pau · Post by **Pau** » Sun Dec 30, 2018 1:06 pm

zzffnn wrote:Even #1 cover slips made in China these days can be pretty good. I measured 10 AmScope #1 cover slips previously, with a micrometer, and found only one 0.15 or 0.14mm (the other 9 were all at 0.17mm).

So they must had been labeled #1.5

Beatsy · Post by **Beatsy** » Sun Dec 30, 2018 5:57 pm

zzffnn wrote:Liquid column depth can be measured by microscope: focus onto top surface of microscope slide, note the fine focus location, move up fine focus into good focus on bottom of cover slip, note find focus location again and calculate that depth difference (which is the liquid column depth).

Don't forget to multiply the measured distance by the RI of the liquid. Not sure, but I think the cover slip RI will affect that measurement too. Don't know how though.

abednego1995 · Post by **abednego1995** » Sun Dec 30, 2018 7:19 pm

Sadly, I did the test 2years ago, and at the time the criteria for testing was only to rule out a faulty objective for a quick return to the seller. I should have kept the slide.

As Beatsy has pointed out, there is little meaning in measuring the depth without knowing the RI of the glass used since measuring the physical depth by focusing will only reveal the total optical path length.

However, how the GAIIx works reveals some answers.
I've already mentioned that the GAIIx is a TIRF(Total Internal Reflection Fluorescence) microscope in principle. TIRF utilizes the evanescent field in the immediate vicinity of a total reflection for excitation of fluorophores. So it only works at the boundary of TIR. Of the many methods of TIRF, the GAIIx works by using a prism oiled to the flow-cell to introduce the excitation laser (in contrary to using high NA objectives and Epi illumination) To achieve TIR, angle of the laser introduced is crucial. It must be over the critical angle dictated by the boundary between the lower inner surface of the flow-cell and the buffer running through the cell. TIR only works at the first RI boundary switch from high>low, So the GAIIx cannot utilize multiple surface excitation and read-out by principle.

Hence, we can come to the conclusion that the objectives meant for the GAIIx should be corrected for observing the lower internal surface of the cell.

quick and dirty schematic

Cheers,
John

Lou Jost · Post by **Lou Jost** » Mon Dec 31, 2018 12:42 pm

This is excellent information, thanks so much! The patent application left open the possibility that either the upper or lower inner surfaces were used.

Is the "evanescent field" due to virtual quantum tunneling based on the Heisenberg uncertainty?

According to the patent, the cell wall thickness is 100um and the inner space is 300um deep. This corresponds to the approx 340-510um optimum measured in my tests, but my accuracy is limited because of the fixed thickness of 0.17mm for my cover slips.

Do you know the refractive index of the liquid used in the sequencer? That would be the last bit of info needed to optimize the cover slip thickness.

Lou Jost · Post by **Lou Jost** » Mon Dec 31, 2018 2:04 pm

There's one other remaining question about these objectives; are they really VC (violet corrected), as the eBay descriptions often claim? Violet correction means that the objective is corrected for the 405nm line as well as the normal three lines of an apo lens. Is there any reason why that correction might be useful in this application? The VC objectives are also supposed to have better resolution in the edges of the field. I suppose that would be important in any high-end application even if the 405nm line was unimportant.

abednego1995 · Post by **abednego1995** » Mon Dec 31, 2018 9:08 pm

Well, actually I've already asked my Nikon rep that question.
Their answer is "No." Though I think this more of a non-disclosure problem.
I can maybe do a quick stack on my cells with a confocal to confirm this, but no, I don't have time for the time being.

Then, naturally we have to go back and examine the instrument.
The GaIIx uses 2 lasers for excitation. 532, 660nm. As fluorescesce will always be at a longer wavelength than excitation(unless you are using multi-photon excitation, which is another story), there is no necessity for using a objective corrected down to 405nm. Of course it will work, but it's not necessary.

Lou Jost · Post by **Lou Jost** » Mon Dec 31, 2018 9:18 pm

That's good information, thanks. No one else has been able to suggest why these would be VC, so maybe that's an internet "enhancement".

abednego1995 · Post by **abednego1995** » Mon Dec 31, 2018 9:22 pm

And, I think the PA20VC and normal PA20 are already rated at OFN25mm at this generation. Are there any literature regarding peripheral resolution for the PA20VC?

P.S. I didn't mention the easiest way to use a high NA 0.17mm coverslip corrected objective on a bare specimen. Just place a high precision 0.170mm coverslip on the tip of the objective. It'll shorten the available WD, but will work charms. Try it on a PA10x. The WD on a PA20x is a bit pain in the ##### for normal macro-photography.

Lou Jost · Post by **Lou Jost** » Mon Dec 31, 2018 9:45 pm

I had found a Nikon pamphlet explaining the advantages of VC, and it included schematic side-by-side fields of view of non-VC and VC objectives, with the VC objective resolving to the edge of the frame. This is where I got my info on the better peripheral performance of VC objectives:
https://www.nikon.com/products/microsco ... _07_lr.pdf

I also found other Nikon brochures which alluded to this when discussing VC objectives:

"The CFI Plan Apochromat VC series Objective Lenses are top performance objectives with perfect correction of chromatic aberrations in the visible light range and excellent resolution throughout the field of view."

"Observation of images with excellent brightness
throughout the view field by minimizing the light loss
around the edges—a critical criterion for digital-image
capturing."

abednego1995 · Post by **abednego1995** » Mon Dec 31, 2018 9:54 pm

Thanks for the info, I think I've seen this in another form. The objective they're using here is a 100x, and yes achieving large flat field at high mags is difficult. So I'm kind of thinking this is some marketing hype. The CFI60 100x has already gone under 2 iterations since this publication in 2005, so I think the newest CFI60 100x (with nano-crystal coating) is at least at par with the PA100xVC (no evidence, just guessing here)

The PA20 should have less vignetting problems than the PA100.

Munich · Post by **Munich** » Tue Jan 01, 2019 1:47 am

a few illuminating (pun intended) illustrations at
https://twitter.com/KKKKosal/status/983942621448949760
comments in Japanese, translate well via Google

ChrisR · Post by **ChrisR** » Tue Jan 01, 2019 4:42 am

I've been looking for (cheap) thick coverslips. Some only come with cheap haemocytometers (which I've tried - they're fine!):

0.5mm x 2 $15 140882710764
0.3mm x 5 similar 281195223587
0.4mm x10 £30 https://www.fishersci.co.uk/shop/produc ... c/10099780
and on Amazon (thickness 0.4mm visible on box label image)

0.4mm x 10 AUD$7 http://www.hurstscientific.com.au/micro ... g2026.html

?? thick, x 100, $15 Canada https://www.abmgood.com/Hemocytometer-G ... .html#faqs

0.4mm x500 $25 https://www.aliexpress.com/item/New-500 ... 17309.html

0.5mm x 100 $12.89 https://www.aliexpress.com/item/New-500 ... 17309.html

The thickness isn't critical for that use, so may not be accurate.

Lou Jost · Post by **Lou Jost** » Tue Jan 01, 2019 6:12 am

Munich, I recognize those figures from the patent application referenced in our original thread...I imagine the text is just a Japanese translation of the English in that application?

https://patents.google.com/patent/US8143599