Quantitative phase and refractive index measurements with point-source digital in-line holographic microscopy.

Point-source digital in-line holographic microscopy with numerical reconstruction is ideally suited for quantitative phase measurements to determine optical path lengths and to extract changes in refractive index within accuracy close to 0.001 on the submicrometer length scale. This is demonstrated with simulated holograms and with detailed measurements on a number of different micrometer-sized samples such as suspended drops, optical fibers, as well as organisms of biological interest such as E. coli bacteria, HeLa cells, and fibroblast cells.

Comment on "Reconstruction algorithm for high-numerical-aperture holograms with diffraction-limited resolution".

We comment on a recent Letter by Zhang et al. [Opt. Lett. 31, 1633 (2006)] in which the authors proposed a reconstruction algorithm for high-numerical-aperture (NA) holograms. Such an algorithm has been available for in-line holography for more than a decade. The authors' "achievement" of high NA for digital in-line holography, NA=0.17, is below what was reported already several years ago (NA=0.30) and is considerably lower than what is routinely achieved now. We present reconstructions of holograms acquired with NAs above 0.4 in which we show maximal achievable resolution.

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.

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 particle's 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.

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 microscope measurements of long-range forces near lipid-coated surfaces in electrolytes.

The interaction of DMPC (L-alpha-dimyristoyl-1,2-diterradecanoyl-sn-glycero-3-phosphoch oli ne, C36H72NO8P) lipid-coated Si3N4 surfaces immersed in an electrolyte was investigated with an atomic force microscope. A long-range interaction was observed, even when the Si3N4 surfaces were covered with nominally neutral lipid layers. The interaction was attributed to Coulomb interactions of charges located at the lipid surface. The experimental force curves were compared with solutions for the linearized as well as with exact solutions of the Poisson-Boltzmann equation. The comparison suggested that in 0.5 mM KCl electrolyte the DMPC lipids carried about one unit of charge per 100 lipid molecules. The presence of this surface charge made it impossible to observe an effective charge density recently predicted for dipole layers near a dielectric when immersed in an electrolyte. A discrepancy between the theoretical results and the data at short separations was interpreted in terms of a decrease in the surface charge with separation distance.

Modeling and measuring the elastic properties of an archaeal surface, the sheath of Methanospirillum hungatei, and the implication of methane production.

We describe a technique for probing the elastic properties of biological membranes by using an atomic force microscope (AFM) tip to press the biological material into a groove in a solid surface. A simple model is developed to relate the applied force and observed depression distance to the elastic modulus of the material. A measurement on the proteinaceous sheath of the archaebacterium Methanospirillum hungatei GP1 gave a Young's modulus of 2 x 10(10) to 4 x 10(10) N/m2. The measurements suggested that the maximum sustainable tension in the sheath was 3.5 to 5 N/m. This finding implied a maximum possible internal pressure for the bacterium of between 300 and 400 atm. Since the cell membrane and S-layer (wall) which surround each cell should be freely permeable to methane and since we demonstrate that the sheath undergoes creep (expansion) with pressure increase, it is possible that the sheath acts as a pressure regulator by stretching, allowing the gas to escape only after a certain pressure is reached. This creep would increase the permeability of the sheath to diffusible substances.

Theory of electrostatic effects in soft biological interfaces using atomic force microscopy.

We calculated the electrostatic force between a planar interface, such as a planar-supported lipid bilayer membrane, and the tip of a stylus on which another lipid bilayer or some other biomacromolecular system might be deposited. We considered styli with rounded tips as well as conical tips. To take into account the effect of dynamical hydrogen-bonded structures in the aqueous phase, we used a theory of nonlocal electrostatics. We used the Derjaguin approximation and identified the systems for which its use is valid. We pointed out where our approach differs from previous calculations and to what extent the latter are inadequate. We found that 1) the nonlocal interactions have significant effects over distances of 10-15 A from the polar zone and that, at the surface of this zone, the effect on the calculated force can be some orders of magnitude; 2) the lipid dipoles and charges are located a distance L from the hydrophobic layer in the aqueous medium and this can have consequences that may not be appreciated if it is ignored; 3) dipoles, located in the aqueous region, can give rise to forces even though the polar layer is unchanged, and if this is ignored the interpretation of force data can be erroneous if an attempt is made to rationalize an observed force with a knowledge of an uncharged surface; 4) the shape of the stylus tip can be very important, and a failure to take this into account can result in incorrect conclusions, a point made by other workers; and 5) when L is nonzero, the presence of charges and dipoles can yield a force that can be nonmonotonic as a function of ionic concentration.

Transmission electron microscopy, scanning tunneling microscopy, and atomic force microscopy of the cell envelope layers of the archaeobacterium Methanospirillum hungatei GP1.

Methanospirillum hungatei GP1 possesses paracrystalline cell envelope components including end plugs and a sheath formed from stacked hoops. Both negative-stain transmission electron microscopy (TEM) and scanning tunneling microscopy (STM) distinguished the 2.8-nm repeat on the outer surface of the sheath, while negative-stain TEM alone demonstrated this repeat around the outer circumference of individual hoops. Thin sections revealed a wave-like outer sheath surface, while STM showed the presence of deep grooves that precisely defined the hoop-to-hoop boundaries at the waveform nodes. Atomic force microscopy of sheath tubes containing entrapped end plugs emphasized the end plug structure, suggesting that the sheath was malleable enough to collapse over the end plugs and deform to mimic the shape of the underlying structure. High-resolution atomic force microscopy has revised the former idea of end plug structure so that we believe each plug consists of at least four discs, each of which is approximately 3.5 nm thick. PT shadow TEM and STM both demonstrated the 14-nm hexagonal, particulate surface of an end plug, and STM showed the constituent particles to be lobed structures with numerous smaller projections, presumably corresponding to the molecular folding of the particle.

High-resolution topography of the S-layer sheath of the archaebacterium Methanospirillum hungatei provided by scanning tunneling microscopy.

The inner and outer surfaces of the sheath of Methanospirillum hungatei GP1 have been imaged for the first time by using a bimorph scanning tunneling microscope (STM) on platinum-coated or uncoated specimens to a nominal resolution in height of ca. 0.4. nm. Unlike more usual types of microscopy (e.g., transmission electron microscopy), STM provided high-resolution topography of the surfaces, giving good depth detail which confirmed the sheath to be a paracrystalline structure possessing minute pores and therefore impervious to solutes possessing a hydrated radius of greater than 0.3 nm. STM also confirmed that the sheath consisted of a series of stacked hoops approximately 2.5 nm wide which were the remnants of the sheath after treatment with 2% (wt/vol) sodium dodecyl sulfate-2% (vol/vol) beta-mercaptoethanol (pH 9.0). No topographical infrastructure could be seen on the sides of the hoops. This research required the development of a new long-range STM capable of detecting small particles such as bacteria on graphite surfaces as well as a new "hopping" STM mode which did not deform the poorly conducting bacterial surface during high-resolution topographical analysis.

Fiber optic displacement sensor with subangstrom resolution.

A simple Fabry-Perot interferometer that uses a singlemode bidirectional fiber coupler as a beam splitter is described. With light from a high stability He-Ne laser focused on the optic fiber, noise levels of 0.5-A rms equivalent reflector displacements were obtained in a bandwidth of 0.02-1 kHz.