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Spatial calibration of structured illumination fluorescence microscopy using capillary tissue phantoms
Author(s) -
Lee Grace S.,
Miele Lino F.,
Turhan Aslihan,
Lin Miao,
Hanidziar Dusan,
Konerding Moritz A.,
Mentzer Steven J.
Publication year - 2009
Publication title -
microscopy research and technique
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 118
eISSN - 1097-0029
pISSN - 1059-910X
DOI - 10.1002/jemt.20647
Subject(s) - confocal microscopy , confocal , microscopy , light sheet fluorescence microscopy , materials science , optical sectioning , biomedical engineering , microscope , optics , microcirculation , calibration , bright field microscopy , resolution (logic) , imaging phantom , fluorescence microscope , intravital microscopy , image resolution , microvessel , scanning confocal electron microscopy , pathology , fluorescence , computer science , physics , medicine , artificial intelligence , immunohistochemistry , quantum mechanics , radiology
Abstract Quantitative assessment of microvascular structure is relevant to the investigations of ischemic injury, reparative angiogenesis and tumor revascularization. In light microscopy applications, thick tissue specimens are necessary to characterize microvascular networks; however, thick tissue leads to image distortions due to out‐of‐focus light. Structured illumination confocal microscopy is an optical sectioning technique that improves contrast and resolution by using a grid pattern to identify the plane‐of‐focus within the specimen. Because structured illumination can be applied to wide‐field (nonscanning) microscopes, the microcirculation can be studied by sequential intravital and confocal microscopy. To assess the application of structured illumination confocal microscopy to microvessel imaging, we studied cell‐sized microspheres and fused silica microcapillary tissue phantoms. As expected, structured illumination produced highly accurate images in the lateral ( X‐Y ) plane, but demonstrated a loss of resolution in the Z‐Y plane. Because the magnitude of Z ‐axis distortion was variable in complex tissues, the silica microcapillaries were used as spatial calibration standards. Morphometric parameters, such as shape factor, were used to empirically optimize Z ‐axis software compression. We conclude that the silica microcapillaries provide a useful tissue phantom for in vitro studies as well as spatial calibration standard for in vivo morphometry of the microcirculation. Microsc. Res. Tech., 2009. © 2008 Wiley‐Liss, Inc.