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.

Cellular motility is an ancient eukaryotic trait, ubiquitous across phyla with roles in predator avoidance, resource access, and competition. Flagellar motility is seen in various parasitic protozoans, and morphological changes in flagella during the parasite life cycle have been observed. We studied the impact of these changes on motility across life cycle stages, and how such changes might serve to facilitate human infection. We used holographic microscopy to image swimming cells of different Leishmania mexicana life cycle stages in three dimensions. We find that the human-infective (metacyclic promastigote) forms display ‘run and tumble’ behaviour in the absence of stimulus, reminiscent of bacterial motion, and that they specifically modify swimming direction and speed to target host immune cells in response to a macrophage-derived stimulus. Non-infective (procyclic promastigote) cells swim more slowly, along meandering helical paths. These findings demonstrate adaptation of swimming phenotype and chemotaxis towards human cells.

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Cellular motility is an ancient eukaryotic trait, ubiquitous across phyla with roles in predator avoidance, resource access and competition. Flagellar-dependent motility is seen in a variety of parasitic protozoans and morphological changes in flagellar structure and function have been qualitatively described during differentiation. However, whether the dynamics of flagellar motion vary across lifecycle stages and whether such changes serve to facilitate human infection is not known. Here we used holographic video microscopy to study the pattern of motility in insect midgut forms of Leishmania (procyclic promastigotes; PCF) and differentiated human infective metacyclic promastigotes (META). We discovered that PCF swim in a slow, corkscrew motion around a gently curving axis while META display run and tumble behaviour in the absence of stimulus, reminiscent of bacterial behaviour. In addition, we demonstrate that META specifically respond to a macrophage-derived stimulus, modifying swimming direction and speed to target host immune cells. Thus, the motility strategy employed by Leishmania appears as a random search that is replaced with a ballistic swimming motion in the presence of an immunological stimulus. These findings shed unique insights into how flagellar motion adapts to the particular needs of the parasite at different times in its lifecycle and define a new pre-adaptation for infection of the human host.

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