Hi there, I’m a hobbyist. More precisely I’m a high-schooler from Austria, about 16 years old, and I have fallen in love with microscopy.
I wanted to ask about how different the Numerical Aperture has an effect on the resolution and brightness of an image. It would be great if somebody could give me visual comparisons. For example, between 1.0 NA and 1.3 NA.
Also, I have also stumbled upon the concept of 40x oil objectives. People frequently tell me how oil improves resolution and brightness. These objectives apparently exist at these low magnifications, and I thought it would be convenient for scanning the slide while simultaneously having the resolution of oil. I want some opinions on this, and it would be great if somebody had any visual comparisons between a dry and oil objective.
Hi there, I hope you continue to enjoy microscopy. There is a whole world out there to explore, both using microscopy in various fields and developing microscopy techniques and tools.
There are analytical expressions for resolution and brightness which involve the NA (assuming ideal optics). Briefly, lateral resolution scales as 1/NA, axial resolution scales as 1/NA^2, and brightness scales as NA^2. So all else equal a 1.3 NA objective compared to an NA 1.0 objective will have lateral resolution of 0.77x (i.e. features closer together can be resolved as separate objects), axial resolution of 0.59x, and brightness improvement of 1.69x.
When people say “oil improves resolution and brightness” they really mean that oil objectives usually have larger NA. It is because of the larger NA that oil objectives offer better resolution and brightness. But there are plenty of intricacies, e.g. biological samples are not usually index-matched to oil so there will be depth-dependent spherical aberration, and those aberrations may or may not matter depending on the use case. Like I said, there’s a whole world to explore…
If you would like an easily accessible visual explanation of these concepts in microscopy you may find my YouTube channel of interest. I mix practical ‘How to build a microscope’ content with visual explanation of theory. Take a look at some of the videos to see if they will help you:
The relation between oil objective, NA and resolution is not always straightforward.
Before jumping to increasing NA, keep in mind that your resolution is first limited by your camera pixel size and your magnification, if you use binoculars or if the pixel size is already small enough (should be ~half of your expected resolution), you will improve resolution by a factor of 1/NA with increasing NA values, up to 1.33. Beyond this you should not see obvious improvement. (because your media is water, light corresponding to NA>1.33 behave differently).
If you need to calculate it… for example with a x100 objective your resolution is ~half of your wavelength divided by NA. If your NA is 1.0 and you use white light, your resolution is ~0.5*550/1 = 225nm. On the camera plane this become 22.5µm (x100) So your camera pixel size should be 22µm or less.
Finally, an oil objective can support NA up to 1.5, so it may offer better resolution. Though in my experience brightness may diminish (they reflect more light than conventional objectives as they have more lenses inside). I’m not sure that the improvement in resolution will be so obvious to the naked eye, compared to a 1.3 NA. Oil is also a bit more bothersome to use.
Agreed: you need to have enough pixels to preserve optical resolution.
I often see mistakes the other way, for instance people erroneously think that a 100x/1.3 oil objective will have better resolution than a 60x/1.4 oil objective. As long as you are adequately sampling – by Nyquist the minimum pixel size 2-fold smaller than optical resolution – then the achieved resolution is determined by the NA.
For example, if your optical resolution is 300nm then your experimental resolution suffers if your pixel size is larger than 150nm. But you don’t get any extra information from having 50nm pixels instead of 100nm pixels (just 4-fold more data on disk) and in fact noise will be worse because each pixel will have fewer photoelectrons. In special situations there can be valid reasons to oversample, but the general point is that you should ensure your pixel size is a “Goldilocks” value, not so large that resolution suffers but also not so small that you are collecting information-poor data.