🔬Microscope Resolution Calculator - Abbe Limit, NA, Useful Magnification, Nyquist

How to use Microscope Resolution & Imaging Calculator

1. 1Choose an objective (dry, water or oil immersion) and wavelength to see the resolution by each criterion
2. 2Drag the separation of two point sources to see when their Airy disks merge
3. 3On the magnification, camera and depth-of-field pages set eyepiece, field number, camera pixel and adapter to check for empty magnification or undersampling
4. 4Compare contrast methods and super-resolution techniques on the illumination and beyond-the-limit pages

FAQ

What is the smallest thing a light microscope can resolve?

Diffraction limits lateral resolution to about d = 0.61λ/NA (Rayleigh). With 550 nm green light and the best oil objective (NA 1.4) that is about 240 nm, and the Abbe limit λ/2NA is about 200 nm. A light microscope shows the outline of bacteria and mitochondria, but not the shape of a virus: a single virus is at best a blurry spot.

Why doesn’t more magnification show more detail?

Detail is set by the objective’s NA; eyepieces and digital zoom only enlarge an image that has already formed. Useful magnification is usually 500×NA to 1000×NA. A 40×/0.65 objective with a 10× eyepiece gives 400×, which is fine; a 25× eyepiece takes it to 1000×, above 650×, which is empty magnification: bigger but blurrier.

Why does oil immersion improve resolution?

NA = n·sinθ. In a dry objective light leaves the specimen through air (n = 1), so even with a collection angle near 72° the NA tops out around 0.95, and steep rays are lost to total internal reflection at the coverslip. Oil with n = 1.518 between coverslip and lens raises n and removes that reflection, allowing NA of 1.4 or more and about 1.5× better resolution.

What pixel size should a microscope camera have?

By Nyquist you need at least 2 (typically 2.3) pixels per resolution element at the specimen. Pixel at the specimen = camera pixel ÷ (objective × adapter magnification). A 6.5 µm pixel with a 60×/1.4 objective and 1× adapter gives 108 nm; for 520 nm emission the resolution is about 227 nm, a ratio near 2.1, which is right. With a 0.5× adapter it would be undersampled.

How do super-resolution microscopes beat the diffraction limit?

They don’t make the spot smaller; they exploit fluorescent molecules that can be switched. STED switches off fluorescence at the rim of the spot with a doughnut beam, giving d = λ/(2NA√(1+I/Is)). STORM and PALM let only a few molecules glow at a time and localize each one to a few to tens of nanometers using photon statistics. SIM shifts high-frequency information in with patterned light for about 2× improvement. Electron microscopes use picometer wavelengths and are limited by lens aberrations to about 0.1 nm.