Fixation protocols for subcellular imaging by synchrotron-based Fourier transform infrared microspectroscopy.
Authors
Gazi, EhsanDwyer, John
Lockyer, Nicholas P
Miyan, J
Gardner, Peter
Hart, Claire A
Brown, Michael D
Clarke, Noel W
Affiliation
School of Chemical Engineering and Analytical Science, The University of Manchester, Manchester M60 1QD, UK. E.Gazi@picr.man.ac.ukIssue Date
2005-01
Metadata
Show full item recordAbstract
Synchrotron-based Fourier transform infrared (SR-FTIR) microspectroscopy is a powerful bioanalytical technique for the simultaneous analysis of lipids, proteins, carbohydrates, and a variety of phosphorylated molecules within intact cells. SR-FTIR microspectroscopy can be used in the imaging mode to generate biospectroscopic maps of the distribution and intensity profiles of subcellular biomolecular domains at diffraction-limited spatial resolution. However, the acquisition of highly spatially resolved IR images of cells is not only a function of instrumental parameters (source brightness, sampling aperture size) but also the cell preparation method employed. Additionally, for the IR data to be biochemically relevant the cells must be preserved in a life-like state without introducing artefacts. In the present study we demonstrate, for the first time, the differences in biomolecular localizations observed in SR-FTIR images of cells fixed by formalin, formalin-critical point drying (CPD), and glutaraldehyde-osmium tetroxide-CPD, using the PC-3 prostate cancer cell line. We compare these SR-FTIR images of fixed cells to unfixed cells. The influence of chemical fixatives on the IR spectrum is discussed in addition to the biological significance of the observed localizations. Our experiments reveal that formalin fixation at low concentration preserves lipid, phosphate, and protein components without significantly influencing the IR spectrum of the cell.Citation
Fixation protocols for subcellular imaging by synchrotron-based Fourier transform infrared microspectroscopy. 2005, 77 (1):18-30 BiopolymersJournal
BiopolymersDOI
10.1002/bip.20167PubMed ID
15558657Type
ArticleLanguage
enISSN
0006-3525ae974a485f413a2113503eed53cd6c53
10.1002/bip.20167