PMC

Illustration of a single parabola flight maneuver during PFCs indicating the different gravity levels

Flowchart illustrating the post-processing steps. Functional blocks of digital hologram autofocusing are shown with green fill color, and phase image registration steps have blue fill color. Average computation times (referenced to t₀) at various checkpoints are also indicated.

(a) Schematic of the microscope with the inset showing off-axis recording where M: Mirror, BS: Beam Splitter, BE: Beam Expander, R: Reference Beam, and O: Object Beam, (b) picture of the microscope on RPM (Media 1), and (c) picture of the experimental rack on plane (microscope is enclosed in the bottom shelf of the rack where highlighted by green dashed lines)

Four frames extracted from DHM Phase and Fluorescence movie (Media 2), top row from 1g control and bottom row from RPM simulated microgravity. Phase images are located at the top respective image in a false color pseudo 3D format, while fluorescence microscopy images are beneath. At every frame, relative time to the control to microgravity transition is indicated with the gravity level

(a) Comparison of several focus criteria in a propagation distance range of 30cm for an experimental hologram of cell. All criteria excluding variance of amplitude (Var) exhibit a multimodal behavior. Zoomed in inset shows the sharpness ambiguity of plotted criteria. (b) Plot and (c) histogram of the experimentally evaluated autofocusing error of ‘Var’ in object space for various axial sample positions with the center being the physical imaging condition (dashed orange lines indicate ± DOF/2).

DHM Phase movie (Media 3) of a cell acquired during ESA 53rd PFC. Left) False color phase image with reference color bar (top) in degrees. Right) Dynamic phase variation plot in a central region of the cell (indicated with dashed circle) with respect to the instantaneous gravity level. Black dashed line indicates to baseline phase value before the parabola set, and recovery to this value occurs at the end of the parabola set break marked by the arrow.

Imaging of living C2C12 cells expressing eGFP tagged actin on the RPM platform (phase and fluorescence). Phase images with (a) no propagation, (b) after autofocusing, and (c) final registration output are shown for the RPM at rest (d) Fluorescence microscopy mode image after hologram acquisition is shown for comparison. (e-l) Phase images constructed from two holograms and corresponding fluorescence images during RPM rotation show the performance of our proposed post-processing. (Scalebars: 10µm). White arrows on the first column of images points to the cell extent where defocus is best observed.