Digital holographic microscope recordings of passive motion of small particles

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.