Signal analysis of total internal reflection fluorescent speckle microscopy (TIR-FSM) and wide-field epi-fluorescence FSM of the actin cytoskeleton and focal adhesions in living cells

M. C. Adams, A. Matov, D. Yarar, S. L. Gupton, G. Danuser, Clare M. Waterman-Storer

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

Fluorescent speckle microscopy (FSM) uses low levels of fluorescent proteins to create fluorescent speckles on cytoskeletal polymers in high-resolution fluorescence images of living cells. The dynamics of speckles over time encode subunit turnover and motion of the cytoskeletal polymers. We sought to improve on current FSM technology by first expanding it to study the dynamics of a non-polymeric macromolecular assembly, using focal adhesions as a test case, and second, to exploit for FSM the high contrast afforded by total internal reflection fluorescence microscopy (TIR-FM). Here, we first demonstrate that low levels of expression of a green fluorescent protein (GFP) conjugate of the focal adhesion protein, vinculin, results in clusters of fluorescent vinculin speckles on the ventral cell surface, which by immunofluorescence labelling of total vinculin correspond to sparse labelling of dense focal adhesion structures. This demonstrates that the FSM principle can be applied to study focal adhesions. We then use both GFP-vinculin expression and microinjected fluorescently labelled purified actin to compare quantitatively the speckle signal in FSM images of focal adhesions and the actin cytoskeleton in living cells by TIR-FM and wide-field epifluorescence microscopy. We use quantitative FSM image analysis software to define two new parameters for analysing FSM signal features that we can extract automatically: speckle modulation and speckle detectability. Our analysis shows that TIR-FSM affords major improvements in these parameters compared with wide-field epifluorescence FSM. Finally, we find that use of a crippled eukaryotic expression promoter for driving low-level GFP-fusion protein expression is a useful tool for FSM imaging. When used in time-lapse mode, TIR-FSM of actin and GFP-conjugated focal adhesion proteins will allow quantification of molecular dynamics within interesting macromolecular assemblies at the ventral surface of living cells.

Original languageEnglish (US)
Pages (from-to)138-152
Number of pages15
JournalJournal of Microscopy
Volume216
Issue number2
DOIs
StatePublished - Nov 2004

Fingerprint

Focal Adhesions
signal analysis
Signal analysis
Speckle
Actin Cytoskeleton
Microscopy
Microscopic examination
adhesion
Adhesion
Fluorescence
Cells
microscopy
fluorescence
Vinculin
proteins
Green Fluorescent Proteins
Proteins
Fluorescence Microscopy
Actins
Polymers

Keywords

  • Cell migration
  • Fluorescent speckle microscopy
  • Total internal reflection FSM

ASJC Scopus subject areas

  • Instrumentation

Cite this

Signal analysis of total internal reflection fluorescent speckle microscopy (TIR-FSM) and wide-field epi-fluorescence FSM of the actin cytoskeleton and focal adhesions in living cells. / Adams, M. C.; Matov, A.; Yarar, D.; Gupton, S. L.; Danuser, G.; Waterman-Storer, Clare M.

In: Journal of Microscopy, Vol. 216, No. 2, 11.2004, p. 138-152.

Research output: Contribution to journalArticle

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abstract = "Fluorescent speckle microscopy (FSM) uses low levels of fluorescent proteins to create fluorescent speckles on cytoskeletal polymers in high-resolution fluorescence images of living cells. The dynamics of speckles over time encode subunit turnover and motion of the cytoskeletal polymers. We sought to improve on current FSM technology by first expanding it to study the dynamics of a non-polymeric macromolecular assembly, using focal adhesions as a test case, and second, to exploit for FSM the high contrast afforded by total internal reflection fluorescence microscopy (TIR-FM). Here, we first demonstrate that low levels of expression of a green fluorescent protein (GFP) conjugate of the focal adhesion protein, vinculin, results in clusters of fluorescent vinculin speckles on the ventral cell surface, which by immunofluorescence labelling of total vinculin correspond to sparse labelling of dense focal adhesion structures. This demonstrates that the FSM principle can be applied to study focal adhesions. We then use both GFP-vinculin expression and microinjected fluorescently labelled purified actin to compare quantitatively the speckle signal in FSM images of focal adhesions and the actin cytoskeleton in living cells by TIR-FM and wide-field epifluorescence microscopy. We use quantitative FSM image analysis software to define two new parameters for analysing FSM signal features that we can extract automatically: speckle modulation and speckle detectability. Our analysis shows that TIR-FSM affords major improvements in these parameters compared with wide-field epifluorescence FSM. Finally, we find that use of a crippled eukaryotic expression promoter for driving low-level GFP-fusion protein expression is a useful tool for FSM imaging. When used in time-lapse mode, TIR-FSM of actin and GFP-conjugated focal adhesion proteins will allow quantification of molecular dynamics within interesting macromolecular assemblies at the ventral surface of living cells.",
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