Practical Limits of Resolution in Confocal and Non-linear Microscopy

Calculated and measured resolution figures are presented for confocal microscopes with different pinhole sizes and for nonlinear (2-photon and second harmonic) microscopes. A modest degree of super-resolution is predicted for a confocal microscope but in practice this is not achievable and confocal fluorescence gives little resolution improvement over widefield. However, practical non-linear microscopes do approach their theoretical resolution and therefore show no resolution disadvantage relative to confocal microscopes in spite of the longer excitation wavelength.