DOI: 10.5061/dryad.9cnp5hqr7
title: Detectability of unresolved particles in off-axis digital holographic microscopy

description: 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.
description: 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 <strong>23</strong>,
 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.
description: # Detectability of unresolved particles in off-axis digital holographic
 microscopy ## Description
 of the data and file structure The datasets include a series of 2048x2048
 pixel tiff files representing a DHM video at approximately 7 frames per
 second. The timestamps file provides exact timestamps for each image. The
 images are raw holograms taken with a "common-mode" off-axis
 DHM. "Flat" indicates a sample chamber of a coverslip + slide
 "Thick" indicates a 0.8 mm gasket between coverslip and slide.
 Illumination wavelength was 405 nm unless specified to be 520 nm. ##
 Code/Software The data were acquired using the software KOALA (LynceeTec).
 Analysis may be performed using any holographic reconstruction software
 that reads tiff files. The package that we used for our published
 reconstructions is a Fiji plug-in available here:
 The following are the datasets   **30 nm
 alumina** Flat slide 2022.07.27 10-57 Thick chamber 2022.07.27 11-14  
 **100 nm alumina** Flat slide 2022.07.27 10-46   **300 nm alumina** Flat
 2022.07.27 10-44 Thick 2022.07.27 11-02   **1 µm Polystyrene beads** *In
 water* Flat 2022.10.05 16-02 Thick 2022.10.05 16-11   *In immersion oil*
 Flat 2022.10.17 12-45 50 nm gold Flat slide 405 nm 2022.08.02 13-56 Flat
 slide 520 nm 2022.08.02 14-37 100 nm lipid vesicles Flat slide 2022.08.02
 13-21 Thick chamber 2022.08.02 13-47   No dye 2022.06.09 13-00 With
 corrole dye 2022.06.09 13-04   200 nm lipid vesicles Flat slide 2022.08.02
 13-17 Thick chamber 2022.08.02 13-40   Unextruded lipid vesicles No dye
 2022.06.09 12-52 With corrole dye 2022.06.09 12-56