dc.contributorHarvard University
dc.contributorUniversidade Estadual Paulista (Unesp)
dc.date.accessioned2015-11-03T18:06:51Z
dc.date.available2015-11-03T18:06:51Z
dc.date.created2015-11-03T18:06:51Z
dc.date.issued2014-02-01
dc.identifierJove-journal Of Visualized Experiments. Cambridge: Journal Of Visualized Experiments, n. 84, 8 p., 2014.
dc.identifier1940-087X
dc.identifierhttp://hdl.handle.net/11449/130305
dc.identifier10.3791/50488
dc.identifierWOS:000348604100002
dc.description.abstractWeakly-scattering objects, such as small colloidal particles and most biological cells, are frequently encountered in microscopy. Indeed, a range of techniques have been developed to better visualize these phase objects; phase contrast and DIC are among the most popular methods for enhancing contrast. However, recording position and shape in the out-of-imaging-plane direction remains challenging. This report introduces a simple experimental method to accurately determine the location and geometry of objects in three dimensions, using digital inline holographic microscopy (DIHM). Broadly speaking, the accessible sample volume is defined by the camera sensor size in the lateral direction, and the illumination coherence in the axial direction. Typical sample volumes range from 200 mu m x 200 mu m x 200 mu m using LED illumination, to 5 mm x 5 mm x 5 mm or larger using laser illumination. This illumination light is configured so that plane waves are incident on the sample. Objects in the sample volume then scatter light, which interferes with the unscattered light to form interference patterns perpendicular to the illumination direction. This image (the hologram) contains the depth information required for three-dimensional reconstruction, and can be captured on a standard imaging device such as a CMOS or CCD camera. The Rayleigh-Sommerfeld back propagation method is employed to numerically refocus microscope images, and a simple imaging heuristic based on the Gouy phase anomaly is used to identify scattering objects within the reconstructed volume. This simple but robust method results in an unambiguous, model-free measurement of the location and shape of objects in microscopic samples.
dc.languageeng
dc.publisherJournal Of Visualized Experiments
dc.relationJove-journal Of Visualized Experiments
dc.relation1.184
dc.rightsAcesso restrito
dc.sourceWeb of Science
dc.subjectBasic Protocol
dc.subjectIssue 84
dc.subjectHolography
dc.subjectDigital inline holographic microscopy (DIHM)
dc.subjectMicrobiology
dc.subjectmicroscopy
dc.subject3D imaging
dc.subjectStreptococcus bacteria
dc.titleDigital Inline Holographic Microscopy (DIHM) of weakly-scattering subjects
dc.typeArtículos de revistas


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