Microscopy

Microscopy Resolution and Image Formation

Two fluorescent emitters can be physically separate but still appear as one blurred spot. Change their separation, wavelength, numerical aperture, and photon number to see how the point-spread function and photon noise determine what the microscope can actually resolve.

Emitter separation d in nm 250

Emission wavelength λ in nm 520

Numerical aperture NA 1.20

Photons per emitter 1200

Background photons per pixel 2.0

Rayleigh criterion 0.61λ/NA

264 nm

Gaussian PSF FWHM

221 nm

Separation / Rayleigh limit

0.95

Interpretation

Strong overlap

True Object Plane

What the Camera Records

Intensity Profile Through Both Emitters

Main ideas A point emitter is recorded as a finite-width point-spread function rather than a mathematical point. Shorter wavelength and larger numerical aperture narrow the PSF. Photon number changes image noise and confidence, but collecting more photons does not make the diffraction-limited PSF itself narrower.