Off-axis digital holographic microscopy (DHM) provides both amplitude and phase images, and so may be used for label-free 3D tracking of micro- and nano-sized particles of different compositions, including biological cells, strongly absorbing particles, and strongly scattering particles. Contrast is provided by differences in either the real or imaginary parts of the refractive index (phase contrast and absorption) and/or by scattering. While numerous studies have focused on phase contrast and improving resolution in DHM, particularly axial resolution, absent have been studies quantifying the limits of detection for unresolved particles. This limit has important implications for microbial detection, including in life-detection missions for space flight. Here we examine the limits of detection of nanosized particles as a function of particle optical properties, microscope optics (including camera well depth and substrate), and data processing techniques and find that DHM provides contrast in both amplitude and phase for unresolved spheres, in rough agreement with Mie theory scattering cross-sections. Amplitude reconstructions are more useful than phase for low-index spheres and should not be neglected in DHM analysis.

These datasets were collected using a common model digital holographic microscope as described in 

J. K. Wallace, S. Rider, E. Serabyn, J. Kuhn, K. Liewer, J. Deming, G. Showalter, C. Lindensmith, and J. Nadeau, Opt Express 23, 17367 (2015).

Sample chambers were either a coverslip on a microscope slide ("flat") or a coverslip on a slide separated by a silicone gasket of thickness 0.8 mm ("thick").

All wavelengths were 405 nm unless specified otherwise.

Frame rate was the maximum frame rate of the system and could be variable, use timestamps file for precision.