Movies of cellular and sub-cellular motion by digital holographic microscopy

Christopher J. Mann, Lingfeng Yu, Myung K. Kim

Research output: Contribution to journalArticle

80 Citations (Scopus)

Abstract

Background: Many biological specimens, such as living cells and their intracellular components, often exhibit very little amplitude contrast, making it difficult for conventional bright field microscopes to distinguish them from their surroundings. To overcome this problem phase contrast techniques such as Zernike, Normarsky and dark-field microscopies have been developed to improve specimen visibility without chemically or physically altering them by the process of staining. These techniques have proven to be invaluable tools for studying living cells and furthering scientific understanding of fundamental cellular processes such as mitosis. However a drawback of these techniques is that direct quantitative phase imaging is not possible. Quantitative phase imaging is important because it enables determination of either the refractive index or optical thickness variations from the measured optical path length with subwavelength accuracy. Digital holography is an emergent phase contrast technique that offers an excellent approach in obtaining both qualitative and quantitative phase information from the hologram. A CCD camera is used to record a hologram onto a computer and numerical methods are subsequently applied to reconstruct the hologram to enable direct access to both phase and amplitude information. Another attractive feature of digital holography is the ability to focus on multiple focal planes from a single hologram, emulating the focusing control of a conventional microscope. Methods: A modified Mach-Zender off-axis setup in transmission is used to record and reconstruct a number of holographic amplitude and phase images of cellular and sub-cellular features. Results: Both cellular and sub-cellular features are imaged with sub-micron, diffraction-limited resolution. Movies of holographic amplitude and phase images of living microbes and cells are created from a series of holograms and reconstructed with numerically adjustable focus, so that the moving object can be accurately tracked with a reconstruction rate of 300ms for each hologram. The holographic movies show paramecium swimming among other microbes as well as displaying some of their intracellular processes. A time lapse movie is also shown for fibroblast cells in the process of migration. Conclusion: Digital holography and movies of digital holography are seen to be useful new tools for visualization of dynamic processes in biological microscopy. Phase imaging digital holography is a promising technique in terms of the lack of coherent noise and the precision with which the optical thickness of a sample can be profiled, which can lead to images with an axial resolution of a few nanometres.

Original languageEnglish (US)
Article number21
JournalBioMedical Engineering Online
Volume5
DOIs
StatePublished - Mar 23 2006
Externally publishedYes

Fingerprint

Holography
Holograms
Motion Pictures
Microscopy
Microscopic examination
Cells
Imaging techniques
Paramecium
Microscopes
Biological Phenomena
Refractometry
Mitosis
Fibroblasts
Noise
CCD cameras
Visibility
Mach number
Numerical methods
Refractive index
Staining and Labeling

ASJC Scopus subject areas

  • Biomedical Engineering
  • Biophysics
  • Radiological and Ultrasound Technology
  • Radiology Nuclear Medicine and imaging
  • Biomaterials
  • Medicine(all)

Cite this

Movies of cellular and sub-cellular motion by digital holographic microscopy. / Mann, Christopher J.; Yu, Lingfeng; Kim, Myung K.

In: BioMedical Engineering Online, Vol. 5, 21, 23.03.2006.

Research output: Contribution to journalArticle

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