High NA objectives

Dear all

I have a very basic question about objective lenses with NA larger than ~1.4: can for instance a 1.49 NA lens gather more light than a 1.4 (1.33 to 1.38) NA lens from a biological sample? To me it seems that this larger area only participate in especial illumination method like TIRF, while no more light from a sample is possible to be observed through these zone, regardless of the NA of lens (except for SAF ring and very few hundreds of nm depth of sample). In general, I am not sure how they can gather more light and generate brighter or higher resolution images; can they? Can they have better resolving power based on their higher NA for example when they are used in trans-illumination, side-illumination (or prism-based TIRF) light-sheet illumination?
Also, what is their critical role (if any) in super resolution microscopy such as PALM/STORM?
My main concern is that these extra NA may compromise image quality and thus the resolution to some extent. (Olympus 1.7NA)
I am sorry if my question looks very basic and stupid.

Well I believe that you are correct, and that the higher NA objectives don’t collect more light. According to this web-page (Leica) an objective has a typical maximum collection angle of about 144 degrees. Doing the math for an oil immersion objective gives an NA of 1.44 as the maximum assuming 1.515 for the refractive index. As you say, you will get additional collection if the dye is within a few hundred nanometers of the interface because it will preferentially emit into the higher index media (SAF), but I think you are asking about ‘deep’ in the sample?

However I think you can get higher resolution, even if you don’t collect more light, because the wavelength of the light will be shorter in the high index media that is used for example by an Olympus 1.7NA. Could this be helpful for PALM/STORM? I think that would depend on what you want to image. I’d say yes if your imaging a sample that is very thin, but maybe no if the sample is thick and you want to image a few microns deep. The distortions due to the refractive index differences are likely to negate any of the gains from the higher NA.

Also, many microscope companies will let you demo their objectives so you can test them and see if they help.

-Hazen

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I would be interested in getting the response from an objective manufacturer. Here is my take, which is slightly different than @Hazen_Babcock.

Higher NA means higher collection angle by definition. The amount of light collected from an isotropic emitter is proportional to NA^2 (e.g. from an ensemble of randomly oriented fluorophores in fluorescence).

Recall that NA = RI*sin(α), so NA < medium refractive index… unless you figure out how to collect light with more than 90° half-angle. The Olympus 1.7 NA uses a special coverslip and special immersion oil with RI > 1.7. The medium with refractive index called out in this equation is the medium the objective is designed for (i.e. the immersion medium for oil objectives).

Objective lenses that have NA/RI ratio anywhere near the maximum of 1.0 (i.e. large collection angles) usually have very short working distance because otherwise they would have a huge front aperture. TIRF lenses fall into that category. I believe than a NA 1.49 TIRF (standard oil) objective really does have a arcsin(1.49/1.5128) ~= 80 degree collection angle, but it has a tiny working distance (BTW the working distance specified is the free working distance besides the coverslip, if one is specified). For TIRF you are imaging right at the coverslip surface anyway so you don’t need more than the 10s of micron WD anyway.

Objective lenses also have transmission curves which tell you how much light gets through. As I understand the main loss mechanism is residual reflections of the AR coatings on internal lens elements (which might be e.g. 1%/surface). It is conceivable that an objective with lower NA but higher transmission might win the competition for collecting the most photons from an identical sample.

To your question of whether having more NA will compromise image quality, I would say “no” but also be aware that objectives have been designed to perform different tasks and use caution when trying to repurpose them. For example, I am aware of several objectives designed for multi-photon imaging where the PSF size on a camera is significantly larger than the NA would predict. For multi-photon imaging this is acceptable, but if you use on a lightsheet system beware. So indeed it is possible for lenses to have intrinsic “underperforming” resolving power relative to the NA-based theoretical limit. I am not aware of any TIRF lenses with this problem. It is also possible for objectives to get damaged or poorly assembled in a way that they don’t achieve anywhere near the theoretical resolving performance.

Different objective lenses have different corrections depending on the target application. Spherical aberrations are the most common type of imaging aberration and occur as you image deeper into a sample that isn’t index-matched with the immersion medium (hence the popularity of silicone oil objectives where the immersion substance better matches the RI of many samples than either water or conventional oil does). Some objective lenses – depending on intended use case and sensitivity to spherical aberration – have correction collars so the user can adjust internal compensation. Some objective lenses have better flat field (plan) correction than others. Some objectives have better chromatic corrections than others (achromat = corrected for 2 colors, fluor = 3 colors, apo = 4 colors). TIRF objectives are usually well-corrected for flatness and chromatically, but multi-photon objectives often do not perform particularly well in either of these categories…

As a final note, I would say to a certain extent you have to try it out yourself to see if a particular objective works in your application. As Hazen said, sometimes you can arrange to test out objectives.

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Thank you Hazen and Jon for your replies and useful information.

I have asked this issue from Nikon and Olympus, they helped a lot, but I still need more clear answer and no one suggested a side by side test (although I believe that side by side comparison would not be very accurate).
I think the problem is that I am talking about biological samples (which I am sure that most of us working with them). Any objective lens can only observe the lights that come out of the sample. The sample medium has RI about n1=1.38, so even an objective lens can gather all 180 degree of emission light (180 degree inside the sample) we can put at most 1.38 on the other side of Snell’s law to calculate the maximum angle of gathering light: 1.38 = n2sin(a)*. Please note that (a) gives us the maximum refraction angle of light from sample to cover slip and oil (except the evanescent field and SAF) which is about 65 degree for normal oil (1.52) and of course is equal to critical angle that we use in TIRF. see
So it seems no matter what lens and what oil you are using, from a biological sample you cannot gather light from larger angle than critical angle. Yes higher NA can collect more light, but here there is no more incoming light to collect; if you are using other samples (n1) this would be different. However, these lenses are usually used for TIRF illumination or at least short WD imaging, so no one can neglect SAF. This is what I think at the moment; am I right?

For resolution and image quality, I want to mention that at least Olympus confirmed that their 1.5 NA lens has much better image quality than 1.7NA in terms of aberration correction.

Is this a theoretical question or you have an application in mind? A few other considerations. If you want maximum brightness then aim for low magnification with high NA. Something like the Nikon 25X 1.1NA water immersion is very good for this or a 40X oil immersion. This MicroscopyU article explains more https://www.microscopyu.com/microscopy-basics/image-brightness.
Consider refractive index matching to your sample. It may be you are better moving away from oil immersion and to water immersion or silicone immersion (Nikon and Olympus have some very nice options) particularly if you are imaging away from the coverslip. These lenses also have correction collars which correct any spherical aberration.

Some of the high NA TIRF lenses you mention have the disadvantage of not being plan so there is roll off towards the edges of the FOV, but that may not be an issue depending on your desired FOV/detector etc. And of course pixel size is important in the trade-off of resolution and sensitivity/SNR. For PALM/STORM this is usually done with 100X TIRF objectives. Nikon have specialised SR versions with better PSF’s. For the best localisation precisions it’s important to not have aberrations and of course to collect the maximum number of photons per pixel. Depending on the system this is usually a pixel size of 100-160 nm. In this context TIRF illumination helps improve the SNR but my understanding is the this is due to having less out of focus light from the sample rather than improving the light gathering power of the objective. But perhaps someone with a more of an optics background than me can say more on that.

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Dear @DanMetcalf ,

First of all, welcome to the Microforum, and thank you for your reply.
Of course I am considering some applications such as SMLM, but this is a general question about the capability of objective with higher than 1.35-1,4 NA. You are right, but I think using low magnification objective is completely different story. By the way, I am not sure someone uses that expensive 25X water immersion without especial need to long working distance.

Edalat

Hi Edalat,
My take is same as you here “No, the effective NA can’t be higher than that of the sample media”, unless you are imaging a few hundreds of nm near the coverslip surface.

What fabulous answers! As kind of mentioned in the other answers, sample prep and RI of mounting and sample are important, and the WD of super high NA objectives do limit to 100nm or less typically. One thing I know the reps (particularly my local Olympus reps, really honest), always remind me of, in addition to the importance of sample prep, is that to harness maximally achievable NA of any objective, you need to fill the back aperture adequately, e.g. just throwing a higher NA modern objective lens onto an older system won’t necessarily give you better performance if you’re starved for photons getting to the sample, I’ve experienced this myself by comparing my current Olympus 40X 1.30 oil to their new XApo 1.40 oil, on a middle aged Yokogawa spinning disk. Great discussion of this topic!

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@Edalat

It seems as if the above explanations are mainly focussed geometrical optics and Snell’s law.

Assume a setup with an oil immersion high NA objective with NA = 1.4, n2 = 1.5
and a biological sample with n1 = 1.3
in transillumination with n0 = 1.0 (and NA_ill = 1.0)

Then according to the Snell’s law
n2 * sin(α2) = n1 * sin(α1) = 1.0 * sin(α0)

The middle medium with n1=1.3 can be skipped in this theoretical consideration because it only creates a parallel ray offset according to geometrical optics and refraction.
[ sin(α1) = 1.0 * sin(α0)/n1 = 1/n1 at α0=90 => sin(α2) = n1 * sin(α1) / n2 = n1/n1/n2 = 1/n2 ]

The theoretical max effective NA would be according to theoretical consideration
NA_max = 1.0 * sin(90) = 1.0

But what you will find in practice is that this setup will deliver brighter images with higher resolution compared to an air objective with NA = 1.0 and similar magnification.

Abbe has explained the microscopic image formation based on diffraction occurring due to object structures. According his explanation “…it is the light-gathering power of the objective, which will affect the brightness of the image and its resolution.”
Higher NA objectives will gather higher diffraction orders which are emerging at higher angles.
The resolution and brightness of an image are increasing with the number of diffraction contributing to image formation.
A high NA oil immersion lens will gather diffraction orders in angle directions which are not contained in the illumination.

At least for transmitted light microscopy this is the work horse explanation.

So

… of course yes.