Reflective polarizers transmit the desired polarization while reflecting the rest. Wire grid polarizers are a common example of this, consisting of many thin wires arranged parallel to each other. The light that is polarized along these wires is reflected, while light that is polarized perpendicular to these wires is transmitted. Other reflective polarizers use Brewster’s angle. Brewster’s angle is a specific angle of incidence under which only s-polarized light is reflected. The reflected beam is s-polarized and the transmitted beam becomes partially p-polarized.

For linearly polarized light with intensity I0, the intensity transmitted through an ideal polarizer, I, can be described by Malus’ law,

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.

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.

While polarizers select certain polarizations of light, discarding the other polarizations, ideal waveplates modify existing polarizations without attenuating, deviating, or displacing the beam. They do this by retarding (or delaying) one component of polarization with respect to its orthogonal component. To help you determine which waveplate is best for your application, read Understanding Waveplates. Correctly chosen waveplates can convert any polarization state into a new polarization state and are most often used to rotate linear polarization, to convert linearly polarized light to circularly polarized light, or vice versa.

Birefringent polarizers rely on the dependence of the refractive index on the polarization of light. Different polarizations will refract at different angles and this can be used to select certain polarizations of light.

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).

Figure 11 shows a photo taken of Edmund Optics Headquarters and the variation in the color of the sky, grass, and foliage from using or not using a polarizer in front of a camera lens. Because electrons in air molecules scatter light in many directions, the appearance of the sky without a polarizer is a lighter shade of blue, as seen in the left image (without polarizer). Additionally, the surface of leaves of trees and on blades of grass are very slightly reflective. Using a polarizer filters out some of the light reflected from these surfaces, darkening the perceived color of these surfaces.

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.

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Where θ is the angle between the incident linear polarization and the polarization axis. We see that for parallel axes, 100% transmission is achieved, while for 90° axes, also known as crossed polarizers, there is 0% transmission. In real-world applications the transmission never reaches exactly 0%, therefore, polarizers are characterized by an extinction ratio, which can be used to determine the actual transmission through two crossed polarizers.

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.

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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.

Understanding and manipulating the polarization of light is crucial for many optical applications. Optical design frequently focuses on the wavelength and intensity of light, while neglecting its polarization. Polarization, however, is an important property of light that affects even those optical systems that do not explicitly measure it. The polarization of light affects the focus of laser beams, influences the cut-off wavelengths of filters, and can be important to prevent unwanted back reflections. It is essential for many metrology applications such as stress analysis in glass or plastic, pharmaceutical ingredient analysis, and biological microscopy. Different polarizations of light can also be absorbed to different degrees by materials, an essential property for LCD screens, 3D movies, and glare-reducing sunglasses.

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?

The analyzer only transmits light that has experienced a specimen-induced phase shift and continues to block all the unaffected light from the source which was originally polarized by the polarizer. If the birefringence of the specimen is known, it can then be used to determine the specimen thickness. If the specimen thickness is known, it can be used to deduce the birefringence of the specimen. A convenient chart used for this purpose is known as the Michel-Levy interference color chart in Figure 14.

The two orthogonal linear polarization states that are most important for reflection and transmission are referred to as p- and s-polarization. P-polarized (from the German parallel) light has an electric field polarized parallel to the plane of incidence, while s-polarized (from the German senkrecht) light is perpendicular to this plane.

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Another characteristic way to see how polarizers reduce reflective glare is by viewing water surfaces. In Figure 7, the surface of the water appears reflective in the left image, obscuring what is below the surface. On the right, however, the rocky debris on the floor of the body of water is much more clearly visible.

Dichroic polarizers absorb a specific polarization of light, transmitting the rest; modern nanoparticle polarizers are dichroic polarizers.

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.

There are also really high NA lenses, like 1.57 100x lenses*. (sorry I read over the olympys 1.7 NA lens mentioned in the first post) I think those can be used to do TIRF in fixed samples, but do require a different material coverslip and hi refractive index oil. I wonder if there are any publication of using these type of lens in SMLM and if there is any benefit of the increased resolution.

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).

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We have one of those but Ive never tested it, and haven’t found a specific usecase for it. If anybody in this thread has some nice examples for use of this lens, it would be appreciated if you could share.

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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?

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.

Once the analyzer has been aligned perpendicularly to the polarizer, an anisotropic, or birefringent, the specimen is placed on the specimen stage. The specimen rotates the polarized light a designated amount, proportional to the specimen thickness (and thus the optical path distance) and the specimen birefringence, before its light reaches the analyzer.

Nice question. An NA higher than the refractive index of your medium will not collect more propagated light. However as you mentioned a NA>n will allow for TIRF/Evanescent wave propagation from your side of the sample, corresponding to high angle propagated light on the imaging and illumination side.

Hot spots are highly reflective portions of a field within a more diffuse reflecting field. In Figure 8, a polarizer is placed in front of the lens of a camera as well as over the light source illuminating the scene to reduce hot spots.

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.

Ring light guides are popular illumination sources due to their even, diffuse illumination. However, glare or reflection of the ring itself may occur. Polarizing the ring light output and the lens separately can reduce these effects, and bring out surface details as seen in Figure 9.

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In a simple polarization microscope system, a linear polarizer is placed in front of a microscope light source, below the specimen stage, to polarize the light entering the system. Another linear polarizer placed above the specimen stage is referred to as an “analyzer,” as this polarizer is rotated to achieve the desired effect when analyzing the sample and while the first polarizer is kept stationary. The analyzer is then rotated such that the polarization planes of the analyzer and polarizer are 90° apart. When this has been achieved, the microscope has a minimum transmission (crossed polarizers); the amount of light transmission will be proportional to the extinction ratio of the polarizer and analyzer.

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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!

Light is an electromagnetic wave, and the electric field of this wave oscillates perpendicularly to the direction of propagation. Light is called unpolarized if the direction of this electric field fluctuates randomly in time. Many common light sources such as sunlight, halogen lighting, LED spotlights, and incandescent bulbs produce unpolarized light. If the direction of the electric field of light is well defined, it is called polarized light. The most common source of polarized light is a laser.

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.

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.

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.

At this point a strange effect occurs: as the law of light propagation work both ways… you can also collect evanescent light from your sample at such high NA. This is named super critical angle (fluroescence) emission or SAF. In practice this means that fluorophores close to the coverslip will yield more light, but not much will change apart from this.

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.

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)

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.

In order to select a specific polarization of light, polarizers are used. Polarizers can be broadly divided into reflective, dichroic, and birefringent polarizers. More detailed information on which type of polarizer is right for your application can be found in our Polarizer Selection Guide.

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…

In Figure 5, a linear polarizer was placed in front of the lens in a machine vision system to remove obfuscating glare such that an electronic chip could be clearly seen. The left image (without polarizer) shows randomly polarized light scattering off of the many glass surfaces between the object and the camera sensor. Much of the chip is obscured by Fresnel reflection of the unpolarized light. The image on the right (with polarizer) shows the chip without glare obscuring any of the object details, allowing the chip to be viewed, analyzed, and measured without obstruction.

Unstressed clear objects between crossed polarizers should yield a completely dark field, however, when internal material stress is present, the localized changes in refractive index rotate the angle of polarization, resulting in transmission variations.

Unpolarized light can be considered a rapidly varying random combination of p- and s-polarized light. An ideal linear polarizer will only transmit one of the two linear polarizations, reducing the initial unpolarized intensity I0 by half,

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

The angular difference between the axes of polarization of the two polarizers is directly related to the amount of overall light attenuation of the set of polarizers. By changing the angle offset, the optical density of the polarizer set can be varied, achieving a similar effect to using a neutral density filter. This ensures that the overall field is evenly illuminated.

Many different types of microscopy techniques such as differential interference contrast (DIC) microscopy utilize polarizers to achieve a variety of effects.

Edmund Optics® offers a wide variety of polarizers, waveplates, polarizing beamsplitters, and other polarization-manipulating optics.

Polarization control is also very important in the chemical, pharmaceutical, and food and beverage industries. Many important organic chemical compounds, such as active pharmaceutical ingredients or sugars, have multiple orientations. The study of molecules with multiple orientations is called stereochemistry.

The same phenomenon can be seen in the Figure 6. In the left image (without polarizer), unpolarized light from the sun is interacting with the windows of the Edmund Optics building and most of this light is reflecting off the windows. In the right image, a polarizing filter has been applied such that the reflected light, rich in one polarization type, is being blocked from the camera sensor and the photographer, using the other polarization type, can see into the building more easily.

I don’t think having a higher NA will degrade your imaging, but it is possible that on the manufacturer side, focusing on achieving higher NA will yield a tradeoff with something such as planeity and field of view size. This follows what @DanMetcalf mentioned.

Implementing polarization control can be useful in a variety of imaging applications. Polarizers are placed over a light source, lens, or both, to eliminate glare from light scattering, increase contrast, and eliminate hot spots from reflective objects. This either brings out more intense color or contrast or helps to better identify surface defects or other otherwise hidden structures.

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 ]

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By cross-polarizing light with two linear polarizers that are oriented perpendicularly, hot spots can be reduced or eliminated altogether.

In amorphous solids such as glass and plastic, stress from temperature and pressure profiles in the material imparts localized variations and gradients in the material properties, making the material birefringent and nonhomogeneous. This can be quantified in transparent objects using the photoelastic effect, as stress and its related birefringence can be measured with polarized light methodologies.

Molecular compounds that have the same type and number of atoms, but different molecular arrangements are called stereoisomers. These stereoisomers are “optically active” and will rotate polarized light in different directions. The amount of rotation is determined by the nature and the concentration of the compound, allowing polarimetry to detect and quantify the concentration of these compounds. This is the premise for identifying which stereoisomer may be present in a sample, which is important because stereoisomers can have vastly different chemical effects. For example, the stereoisomer limonene is the chemical that gives oranges and lemons their characteristic scents.

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.