Enhanced sensitivity in dark-field microscopy by optimizing the illumination angle.

Dark-field microscopy is a well-known technique used to exclude the bright background of unscattered photons from a measurement. We show that by choosing an appropriate illumination angle, the background of unwanted scattered light can also be suppressed. The collected flux of scattered photons is calculated in the Mie scattering regime for various particle sizes and objectives over a range of illumination angles. In the case that the dark-field measurement is limited by background scattering, we find that the sensitivity can be improved by lowering the objective numerical aperture. The collected photon flux is calculated for an exemplary dark-field microscopy experiment in which lipid granules were studied within yeast cells. Our model suggests that the signal-to-noise ratio was over three-orders-of-magnitude higher than it would have been with an equivalent bright-field setup.