In recent years, in-line holography as originally proposed by Gabor, supplemented with numerical reconstruction, has been perfected to the point at which wavelength resolution both laterally and in depth is routinely achieved with light by using digital in-line holographic microscopy (DIHM). The advantages of DIHM are: (1) simplicity of the hardware (laser- pinhole-CCD camera), (2) magnification is obtained in the numerical reconstruction, (3) maximum information of the 3-D structure with a depth of field of millimeters, (4) changes in the specimen and the simultaneous motion of many species, can be followed in 4-D at the camera frame rate. We present results obtained with DIHM in biological and microfluidic applications. By taking advantage of the large depth of field and the plane-to-plane reconstruction capability of DIHM, we can produce 3D representations of the paths followed by micron-sized objects such as suspensions of microspheres and biological samples (cells, algae, protozoa, bacteria). Examples from biology include a study of the motion of bacteria in a diatom and the track of algae and paramecium. In microfluidic applications we observe micro-channel flow, motion of bubbles in water and evolution in electrolysis. The paper finishes with new results from an underwater version of DIHM.
Digital In-line Holographic Microscopy (DIHM) is a technique that provides depth and lateral resolution of the order of the wavelength throughout a volume of several cubic centimeters for visible light. This outstanding characteristic is reached by means of a simple optical setup and numerical reconstruction of the recorded holograms. It makes DIHM the right tool for applications in many microscopic studies. In this paper we study microfluidic phenomena by means of DIHM. To this end we seed a fluid with micron-size trackers (latex microspheres) and follow their displacement within an observation volume. We apply this technique to several situations such as the flow around a big sphere, flow through microchannels, bubbles in a fluid, bacterial motion in a diatom and the swimming behavior of paramecia and algae in water. By taking advantage in DIHM of the plane-to-plane reconstruction through a large depth of field, we generate 3D renderings of the paths followed by the trackers to
produce a complete picture of the flow pattern, i.e. streamlines and velocity fields.
Digital in-line holographic microscopy (DIHM) can achieve wavelength resolution both laterally and in depth with the simple optical setup consisting of a laser illuminating a wavelength-sized pinhole and a CCD camera for recording the hologram. The reconstruction is done numerically on the basis of the Kirchhoff-Helmholtz transform which yields a three-dimensional image of the objects throughout the sample volume.
Resolution in DIHM depends on several controllable factors or parameters: (1) pinhole size controlling spatial coherence, (2) numerical aperture given by the size and positioning of the recording CCD chip, (3) pixel density and dynamic range controlling fringe resolution and noise level in the hologram and (4) wavelength. We present a detailed study of the individual and combined effects of these factors by doing an analytical analysis coupled with numerical simulations of holograms and their reconstruction. The result of this analysis is a set of criteria, also in the form of graphs, which can be used for the optimum design of the DIHM setup. We will also present a series of experimental results that test and confirm our theoretical analysis. The ultimate resolution to date is the imaging of the motion of submicron spheres and bacteria, a few microns apart, with speeds of hundreds of microns per second.
Using holographic microscopy we have been able to visualize submicron-sized. bacteria in-vivo. A simple holographic method enables us to capture as a single data set the trajectories of micron size objects suspended in water. By subtracting consecutive holograms of a particle suspension and then adding these difference holograms, a final data set is constructed that contains the time evolution of the particle trajectories free from spurious background interference effects. Temporal and spatial resolution at the sub-second and sub-micron levels can easily be achieved. The method is illustrated by recording the motion in 3-D of 5μm diameter latex spheres subject to gravity and electrostatic fields to visualize their micro-fluidic flow. Another example is the 3-D motion of a collection of algae, protozoa and bacteria in water.
An update is given of recent advances in digital in-line holography with numerical reconstruction. It is shown that lateral resolution in the submicron regime can now be achieved routinely and that depth resolution is improved to the point that tracking of submicron particles is feasible in three dimensions.
Digital in-line holography with numerical reconstruction has been developed in our group into a tool that routinely achieves both lateral and depth resolution at and below the micron level. 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 is demonstrated for such diverse objects as fibers, suspensions of microspheres and biological samples (epithelial cells, the eye of Drosophila melanogaster larva), and the advantages are discussed by comparing the holographic reconstructions with images taken with conventional optical microscopy.
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