HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ringkob, T. P. Articles by Greaser, M. L. Search for Related Content PubMed PubMed Citation Articles by Ringkob, T. P. Articles by Greaser, M. L.

Light microscopy and image analysis of thin filament lengths utilizing dual probes on beef, chicken, and rabbit myofibrils¹

T. P. Ringkob^*,2, D. R. Swartz and M. L. Greaser

^* Department of Animal Biotechnology, University of Nevada, Reno 89557; and Department of Anatomy, Indiana University Medical School, Indianapolis 46202; and and Muscle Biology Laboratory, University of Wisconsin, Madison 53706

² Correspondence—phone: 775-784-1628; fax: 775-784-1375; e-mail: tringkob{at}scs.unr.edu.

Abstract

Image analysis procedures for immunofluorescence microscopy were developed to measure muscle thin filament lengths of beef, rabbit, and chicken myofibrils. Strips of beef cutaneous trunci, rectus abdominis, psoas, and masseter; chicken pectoralis; and rabbit psoas muscles were excised 5 to 30 min postmortem. Fluorescein phalloidin and rhodamine myosin subfragment-1 (S1) were used to probe the myofibril structure. Digital images were recorded with a cooled charge-coupled device controlled with IPLab Spectrum software (Signal Analytics Corp.) on a Macintosh operating system. The camera was attached to an inverted microscope, using both the phase-contrast and fluorescence illumination modes. Unfixed myofibrils incubated with fluorescein phalloidin showed fluorescence primarily at the Z-line and the tips of the thin filaments in the overlap region. Images were processed using IPLab and the National Institutes of Health’s Image software. A region of interest was selected and scaled by a factor of 18.18, which enlarged the image from 11 pixels/µm to approximately 200 pixels/µm. An X-Y plot was exported to Spectrum 1.1 (Academic Software Development Group), where the signal was processed with a second derivative routine, so a cursor function could be used to measure length. Fixation before phalloidin incubation resulted in greatest intensity at the Z lines but a more-uniform staining over the remainder of the thin filament zone. High-resolution image capture and processing showed that thin filament lengths were significantly different (P < 0.01) among beef, rabbit, and chicken, with lengths of 1.28 to 1.32 µm, 1.16 µm, and 1.05 µm, respectively. Measurements using the S1 signal confirmed the phalloidin results. Fluorescent probes may be useful to study sarcomere structure and help explain species and muscle differences in meat texture.

Key Words: Actin • Beef • Image Analysis • Immunofluorescence • Microfilaments

This article has been cited by other articles:

				A. D. Weaver, B. C. Bowker, and D. E. Gerrard Sarcomere length influences {micro}-calpain-mediated proteolysis of bovine myofibrils J Anim Sci, June 1, 2009; 87(6): 2096 - 2103. [Abstract] [Full Text] [PDF]

				A. D. Weaver, B. C. Bowker, and D. E. Gerrard Sarcomere length influences postmortem proteolysis of excised bovine semitendinosus muscle J Anim Sci, August 1, 2008; 86(8): 1925 - 1932. [Abstract] [Full Text] [PDF]