Hello,
I am working with a cytigenetic lab, analysing Chromosomes with brightfield microscopes. They just moved from Halogen to LED microsocpes and the team is very uncomfortable with the image. They think that getting a new 100X 1.4NA objectives instead of the 1.35NA objectives they have - will make the difference.
For such samples - will it have a visible effect on the resolution (through the eyepieces). Don’t such biological samples has RI of max 1.38 anyway? The 100X 1.4 cost 3 times the cost of the microscopes…
Hello @Anatka and welcome to the forum.
It is not clear from your message what problem you are trying to correct.
From what you say the only problem is that there is some ‘discomfort’ with the image from new microscopes but the only difference you mention is that they use LED instead of halogen and I don’t see how that has anything to do with the objective lens at all, let alone its NA.
Perhaps you can be more specific about the problem you are trying to fix? Perhaps show us an example image from the old microscope and the new microscope on the same (or same kind of) specimen and then tell use what you consider to be wrong with the new image?
Tell us what make/model and spec of objective was used in the previous (satisfactory) microscope and tell what make/model and specs are used in the objective of the new (uncomfortable) microscope.
If you provide this level of detail, someone here might be able to help.
P
Hi Tadrous,
Thank you for your input. The sample is chromosome for cytology analysis. Using Giemsa stainig. In the old - Zeiss microscope (I think Axioplan 2), using Apo 1.4NA, the lab adds an interference-green filter to add green background which helps to identify minor abberations in the chromosomes. Moving to a new model with Apo 1.35NA and LED, using the same Interference-green filter, the details are not as easy to identify.
The question is - investing and getting a new 1.45NA (no 1.4NA is available I was told) for the enw microscopes will solve the issue, or since moving from Halogen to LED will anyway look so much different - it is a really green background now - that there is some protocol adjustment needed.
Any input will help
If it’s just a question of the colour contrast not being ‘as good’ as with the halogen, and not an issue with resolution of the bands, then I don’t see how a higher NA objective will help. However, if the bands are genuinely harder to resolve then the extra NA of the new objective might help.
It would seem reasonable to try some different green-ish filters first and see of that makes the contrast better. That would be a lot cheaper - only cost a few £$ for some cheap Cokin-type / Generic plastic ‘gel’ filters to try, compared to spending £$1000’s for a new objective. If that doesn’t help then maybe the newer objective idea can be reconsidered.
White LEDs tend to have more blue in their spectrum compared to halogen so a more yellowish-green filter may give you closer colour contrast to what you had before. But I am only guessing here since I still have no idea what the problem actually looks like - just going on my understanding of your descriptions.
P
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HI @Anatka
As mentioned by @P_Tadrous , i don’t think the issue you are observing is due to NA, especially if it worked before, and if seen through eyepiece. The 0.05 change is small, and will mostly have an impact i you use TIRF illumination, but not in your case of straightforward collimated illumination.
The most likely cause is indeed that the new LED illumination provides subjectively different results. Just in case you can double check the green filter orientation, and that the new objective is not damaged/does not have significant aberrations.
I would advise if possible to go through it one element at a time: is it possible to test the previous Apo 1.4NA to compare ? Is it possible to borrow a 1.45 NA objective somewhere before investing in 10k$ … Is it possible to try the previous light illumination on the new system ?
If you have images of before/after it could also help to better target the issue, good luck !
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Another point to take into account is that you need to ensure that your new illumination system is providing sufficient NA of illumination to match or exceed your objective NA rating.
So, if it is truly resolution that is the problem (as opposed to optimal colour contrast) then check that your condenser can provide the required 1.35 NA (at least) otherwise you won’t be using the full 1.35 NA of acceptance of your objective and you will get lower resolution.
My understanding is that most dioptric condensers require oil immersion contact with the back of your slide to achieve this, so if you don’t use that immersion contact between the slide and the condenser condenser (not just between slide and objective) you will not be using the full resolution of the objective. Check the specs of your condenser and how it is meant to be used (some condensers are meant to be used dry up to 0.95 NA and oiled thereafter for higher NA). Obviously, don’t oil any optics (condenser or objective) that are not designed to be oiled or you will damage them - hence check the specs and manual first.