Fixation protocols for subcellular imaging by synchrotron-based Fourier transform infrared microspectroscopy.

2.50
Hdl Handle:
http://hdl.handle.net/10541/76840
Title:
Fixation protocols for subcellular imaging by synchrotron-based Fourier transform infrared microspectroscopy.
Authors:
Gazi, Ehsan; Dwyer, John; Lockyer, Nicholas P; Miyan, J; Gardner, Peter; Hart, Claire A; Brown, Michael D; Clarke, Noel W ( 0000-0001-7776-8059 )
Abstract:
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.
Affiliation:
School of Chemical Engineering and Analytical Science, The University of Manchester, Manchester M60 1QD, UK. E.Gazi@picr.man.ac.uk
Citation:
Fixation protocols for subcellular imaging by synchrotron-based Fourier transform infrared microspectroscopy. 2005, 77 (1):18-30 Biopolymers
Journal:
Biopolymers
Issue Date:
Jan-2005
URI:
http://hdl.handle.net/10541/76840
DOI:
10.1002/bip.20167
PubMed ID:
15558657
Type:
Article
Language:
en
ISSN:
0006-3525
Appears in Collections:
All Christie Publications ; All Paterson Institute for Cancer Research

Full metadata record

DC FieldValue Language
dc.contributor.authorGazi, Ehsan-
dc.contributor.authorDwyer, John-
dc.contributor.authorLockyer, Nicholas P-
dc.contributor.authorMiyan, J-
dc.contributor.authorGardner, Peter-
dc.contributor.authorHart, Claire A-
dc.contributor.authorBrown, Michael D-
dc.contributor.authorClarke, Noel W-
dc.date.accessioned2009-08-10T17:25:46Z-
dc.date.available2009-08-10T17:25:46Z-
dc.date.issued2005-01-
dc.identifier.citationFixation protocols for subcellular imaging by synchrotron-based Fourier transform infrared microspectroscopy. 2005, 77 (1):18-30 Biopolymersen
dc.identifier.issn0006-3525-
dc.identifier.pmid15558657-
dc.identifier.doi10.1002/bip.20167-
dc.identifier.urihttp://hdl.handle.net/10541/76840-
dc.description.abstractSynchrotron-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.en
dc.language.isoenen
dc.subject.meshAnimals-
dc.subject.meshCell Line-
dc.subject.meshGlutaral-
dc.subject.meshMicroscopy, Electron-
dc.subject.meshSpectroscopy, Fourier Transform Infrared-
dc.subject.meshSubcellular Fractions-
dc.subject.meshSynchrotrons-
dc.titleFixation protocols for subcellular imaging by synchrotron-based Fourier transform infrared microspectroscopy.en
dc.typeArticleen
dc.contributor.departmentSchool of Chemical Engineering and Analytical Science, The University of Manchester, Manchester M60 1QD, UK. E.Gazi@picr.man.ac.uken
dc.identifier.journalBiopolymersen
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