Simultaneous two-color imaging in digital holographic microscopy

Abstract

Abstract:We demonstrate the use of two-color digital holographic microscopy (DHM) for imaging microbiological subjects. The use of two wavelengths significantly reduces artifacts present in the reconstructed data, allowing us to image weakly-scattering objects in close proximity to strongly-scattering objects. We demonstrate this by reconstructing the shape of the flagellum of a unicellular eukaryotic parasite Leishmania mexicana in close proximity to a more strongly-scattering cell body. Our approach also yields a reduction of approximately one third in the axial position uncertainty when tracking the motion of swimming cells at low magnification, which we demonstrate with a sample of Escherichia coli bacteria mixed with polystyrene beads. The two-wavelength system that we describe introduces minimal additional complexity into the optical system, and provides significant benefits.

High speed, three-dimensional imaging reveals chemotactic behavior specific to human-infectiveLeishmaniaparasites

“…It therefore seems likely that the run and tumble behaviour observed here originates from a different imperative serving a distinct function specific to Leishmania. We note that foraging strategies among larger animals follow superficially similar While holography under high magnification can reveal the geometry of eukaryotic flagella [15,21], the flagellar beat frequency can be inferred even from low-magnification (i.e. large-scale) tracking data.…”

“…These sequences of images were calculated using the Rayleigh-Sommerfeld back-propagation scheme [34]. We localised the cells using a method based on the Gouy phase anomaly, as described in more detail elsewhere [21,35]. This method segments features based on axial optical intensity gradients within a sample, allowing us to extract three-dimensional coordinates for individual cells in each frame.…”

Haloarchaea swim slowly for optimal chemotactic efficiency in low nutrient environments

“…The stack of numerically refocused images was calculated using the Rayleigh-Sommerfeld back-propagation scheme 49 . We localised the cells 50 using a method based on the Gouy phase anomaly, described in more detail elsewhere 46 , 51 . This method segments features based on axial optical intensity gradients within a sample, allowing us to extract 3D coordinates for individual cells in each frame.…”

Multifocal imaging for precise, label-free tracking of fast biological processes in 3D

“…Many eukaryotic cells, such as sperm or green algae, deploy flagella, lash-like appendages protruding from the cell surface. The 3D flagellar beat propels the cell on a 3D swimming path 23,25 . Because the beat frequency is high (up to 100 Hz) 6,7 , the precise reconstruction of flagellar beating at high temporal and spatial resolution is challenging.…”