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Digital holographic microscope recordings of passive motion of small particles


Lindensmith, Christian; Nadeau, Jay; Rouzie, Devan (2021), Digital holographic microscope recordings of passive motion of small particles, Dryad, Dataset,


Digital holographic microscopy provides the ability to observe throughout a volume that is large compared to its resolution without the need to actively refocus to capture the entire volume. This enables simultaneous observations of large numbers of small objects within such a volume. We have constructed a microscope that can observe a volume of 0.4 x 0.4 x 1.0 µm with submicrometer resolution (in xy) and 2 µm resolution (in z) for observation of microorganisms and minerals in liquid environments on Earth and on potential planetary missions. Because environmental samples are likely to contain mixtures of inorganics and microorganisms of comparable sizes near the resolution limit of the instrument, discrimination between living and non-living objects may be difficult. The active motion of motile organisms can be used to readily distinguish them from non-motile objects (live or inorganic), but additional methods are required to distinguish non-motile organisms and inorganic objects that are of comparable size but different composition and structure. We demonstrate the use of passive motion to make this discrimination by evaluating diffusion and buoyancy characteristics of cells, styrene beads, alumina particles, and gas-filled vesicles of micron scale in the field of view.


Holographic microscopy was performed using a custom off-axis common path instrument as described previously [24]. Briefly, the paired objective lenses had a numerical aperture (NA) of 0.3, yielding an effective magnification of 19.6x. The wavelength used was 405 nm, supplied by a diode laser (Thorlabs S1FC405); at this wavelength, the microscope’s lateral spatial resolution as measured with an Air Force test target was 0.8 µm. Samples for recording were diluted into a 1.0 mm deep chamber having both a reference channel (filled with medium or H2O) and a sample channel. The chamber was bounded by a microscope slide and coverslip.

Data acquisition was performed using a custom open-source software package, DHMx [29]. All recordings were performed at a frame rate of 15 fps for a total of 20-60 s per recording. Reconstruction in amplitude and/or phase was performed using the angular spectrum method [30] using custom Fiji plug-ins as previously described [31]. Phase reconstructions made use a reference hologram to remove noise [32]; this reference hologram was the median of all holograms in the recording. The lateral field of view was 356 x 365 µm for 2048 x 2048 pixels, and the axial z-spacing was chosen to be 1-2 µm based upon the particle size and nominal axial resolution of the system of 2 µm.


Jet Propulsion Laboratory, Award: none

National Science Foundation United States, Award: 1828793