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 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.
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.
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.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.