M. H. Jericho

Digital in-line holographic microscopy

We first briefly review the state of the art of digital in-line holographic microscopy (DIHM) with numerical reconstruction and then discuss some technical issues, such as lateral and depth resolution, depth of field, twin image, four-dimensional tracking, and reconstruction algorithm. We then present a host of examples from microfluidics and biology of tracking the motion of spheres, algae, and bacteria. Finally, we introduce an underwater version of DIHM that is suitable for in situ studies in an ocean environment that show the motion of various plankton species.

Digital in-line holography for biological applications

Digital in-line holography with numerical reconstruction has been developed into a new tool, specifically for biological applications, that routinely achieves both lateral and depth resolution, at least at the micron level, in three-dimensional imaging. The experimental and numerical procedures have been incorporated into a program package with a very fast reconstruction algorithm that is now capable of real-time reconstruction. This capability is demonstrated for diverse objects, such as suspension of microspheres and biological samples (diatom, the head of Drosophila melanogaster), and the advantages are discussed by comparing holographic reconstructions with images taken by using conventional compound light microscopy.

Atomic force microscopy of cell growth and division in Staphylococcus aureus

The growth and division of Staphylococcus aureus was monitored by atomic force microscopy (AFM) and thin-section transmission electron microscopy (TEM). A good correlation of the structural events of division was found using the two microscopies, and AFM was able to provide new additional information. AFM was performed under water, ensuring that all structures were in the hydrated condition. Sequential images on the same structure revealed progressive changes to surfaces, suggesting the cells were growing while images were being taken. Using AFM small depressions were seen around the septal annulus at the onset of division that could be attributed to so-called murosomes (Giesbrecht et al., Arch. Microbiol. 141:315-324, 1985). The new cell wall formed from the cross wall (i.e., completed septum) after cell separation and possessed concentric surface rings and a central depression; these structures could be correlated to a midline of reactive material in the developing septum that was seen by TEM. The older wall, that which was not derived from a newly formed cross wall, was partitioned into two different surface zones, smooth and gel-like zones, with different adhesive properties that could be attributed to cell wall turnover. The new and old wall topographies are equated to possible peptidoglycan arrangements, but no conclusion can be made regarding the planar or scaffolding models.

Tracking particles in four dimensions with in-line holographic microscopy.

We describe a simple holographic method that has enabled us to capture as a single data set the trajectories of micrometer-sized objects suspended in water. By subtracting consecutive holograms of a particle suspension and then adding these difference holograms, we constructed a final data set that contains the time evolution of the particle trajectories free from spurious background interference effects. The method is illustrated by a recording of the motion of 5-10-microm diameter algae in water.

Digital in-line holography of microspheres.

We have used digital in-line holography (DIH) with numerical reconstruction to image micrometer-sized latex spheres as well as ferrimagnetic beads suspended in gelatin. We have examined in detail theoretically and experimentally the conditions necessary to achieve submicrometer resolution of holographic reconstructions. We found that both transparent and opaque particles could be imaged with a resolution that was limited only by the wavelength of the light used. Simple inspection of intensity profiles through a particle allowed an estimate to be made of the particles three position coordinates within an accuracy of a few hundred nanometers. When the derivative of a second-order polynomial fitted to the intensity profiles was taken, the X, Y, Z position coordinates of particles could be determined within +/-50 nm. More-accurate positional resolution should be possible with the help of more-advanced computer averaging techniques. Because a single hologram can give information about a large collection of distributed particles, DIH offers the prospect of a powerful new tool for three-dimensional tracking of particles.

Immersion digital in-line holographic microscopy.

Digital in-line holographic microscopy is a promising new tool for high resolution imaging. We demonstrate, by using latex beads, that a considerable increase in numerical aperture, and, therefore, resolution can be achieved if the space between a source and a CCD camera chip is filled with a high refractive index medium. The high refractive index medium implies a shorter effective wavelength so that submicrometer resolution can be obtained with laser light in the visible range.

Nanoscale Characterization and Determination of Adhesion Forces of Pseudomonas aeruginosa Pili by Using Atomic Force Microscopy

Type IV pili play an important role in bacterial adhesion, motility, and biofilm formation. Here we present high-resolution atomic force microscopy (AFM) images of type IV pili from Pseudomonas aeruginosa bacteria. An individual pilus ranges in length from 0.5 to 7 microm and has a diameter from 4 to 6 nm, although often, pili bundles in which the individual filaments differed in both length and diameter were seen. By attaching bacteria to AFM tips, it was possible to fasten the bacteria to mica surfaces by pili tethers. Force spectra of tethered pili gave rupture forces of 95 pN. The slopes of force curves close to the rupture force were nearly linear but showed little variation with pilus length. Furthermore, force curves could not be fitted with wormlike-chain polymer stretch models when using realistic persistence lengths for pili. The observation that the slopes near rupture did not depend on the pili length suggests that they do not represent elastic properties of the pili. It is possible that this region of the force curves is determined by an elastic element that is part of the bacterial wall, although further experiments are needed to confirm this.

Submersible digital in-line holographic microscope

Few instruments exist that can image microscopic marine organisms in their natural environment so that their locomotion mechanisms, feeding habits, and interactions with surfaces, such as biofouling, can be investigated in situ. We describe here the design and performance of a simple submersible digital in-line holographic microscope that can image organisms and their motion with micron resolution and that can be deployed from small vessels. Holograms and reconstructed images of several microscopic aquatic organisms were successfully obtained down to a depth of 20m. Important microscope characteristics such as the effect of camera pixel size on lateral and depth resolutions as well as the maximum sample volume that can be imaged with a given resolution are discussed in detail.

Swimming speed of three species of Alexandrium (Dinophyceae) as determined by digital in-line holography

N.I. Lewis, W. Xu, S.K. Jericho, H.J. Kreuzer, M.H. Jericho and A.D. Cembella. 2006. Swimming speed of three species of Alexandrium (Dinophyceae) as determined by digital in-line holography. Phycologia 45: 61–70. DOI: 10.2216/04-59.1 Digital in-line holographic (DIH) microscopy was used to track motility in several related species of the marine dinoflagellate Alexandrium in response to temperature after acclimation at selected temperatures. Numerical reconstruction of DIH holograms yielded high-contrast three-dimensional images of the trajectories of many motile cells swimming simultaneously throughout the sample volume. Swimming speed and trajectory were determined for clonal isolates of A. ostenfeldii, A. minutum and A. tamarense within the temperature range from 8 to 24°C. The strains of these species revealed differences in temperature optima for growth and tolerance that were a function of both acclimation responses and genetic factors reflecting the origin of the isolates. The fastest swimming speeds were recorded at 24°C for cells of A. minutum. Acclimated strains of all three species swam significantly slower at lower temperatures, although fastest swimming speeds did not always occur at temperature optima for growth. Aged cells from stationary phase cultures swam more slowly than cells in exponential growth phase. Doublets from a rapidly dividing culture swam faster than singlets from the same culture, confirming the propulsive advantage of paired cells. Holographic microscopy is a powerful tool for the acquisition of detailed observations of swimming behaviour of microalgal cells in the form of three-dimensional trajectories over the appropriate temporal (sub-second) and spatial (micrometer) scales.

Contribution of Type IV Pili to the Virulence of Aeromonas salmonicida subsp. salmonicida in Atlantic Salmon (Salmo salar L.)

ABSTRACT Aeromonas salmonicida subsp. salmonicida, a bacterial pathogen of Atlantic salmon, has no visible pili, yet its genome contains genes for three type IV pilus systems. One system, Tap, is similar to the Pseudomonas aeruginosa Pil system, and a second, Flp, resembles the Actinobacillus actinomycetemcomitans Flp pilus, while the third has homology to the mannose-sensitive hemagglutinin pilus of Vibrio cholerae. The latter system is likely nonfunctional since eight genes, including the gene encoding the main pilin subunit, are deleted compared with the orthologous V. cholerae locus. The first two systems were characterized to investigate their expression and role in pathogenesis. The pili of A. salmonicida subsp. salmonicida were imaged using atomic force microscopy and Tap- and Flp-overexpressing strains. The Tap pili appeared to be polar, while the Flp pili appeared to be peritrichous. Strains deficient in tap and/or flp were used in live bacterial challenges of Atlantic salmon, which showed that the Tap pilus made a moderate contribution to virulence, while the Flp pilus made little or no contribution. Delivery of the tap mutant by immersion resulted in reduced cumulative morbidity compared with the cumulative morbidity observed with the wild-type strain; however, delivery by intraperitoneal injection resulted in cumulative morbidity similar to that of the wild type. Unlike the pili of other piliated bacterial pathogens, A. salmonicida subsp. salmonicida type IV pili are not absolutely required for virulence in Atlantic salmon. Significant differences in the behavior of the two mutant strains indicated that the two pilus systems are not redundant.