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Deconvolution of Calcium Fluorescent Indicator Signal from AFM Cantilever Reflection

Published online by Cambridge University Press:  30 July 2012

G. Monserratt Lopez-Ayon ,
David J. Oliver ,
Peter H. Grutter  and
Svetlana V. Komarova
G. Monserratt Lopez-Ayon*
Affiliation:
Center for the Physics of Materials and the Department of Physics, McGill University, 3600 University, Montreal, Quebec H3A 2T8, Canada
David J. Oliver
Affiliation:
Center for the Physics of Materials and the Department of Physics, McGill University, 3600 University, Montreal, Quebec H3A 2T8, Canada
Peter H. Grutter
Affiliation:
Center for the Physics of Materials and the Department of Physics, McGill University, 3600 University, Montreal, Quebec H3A 2T8, Canada
Svetlana V. Komarova
Affiliation:
Shriners Hospital for Children, Montreal, Quebec, Canada Faculty of Dentistry, McGill University, Montreal, Quebec, Canada
*
Corresponding author. E-mail: [email protected]
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Abstract

Atomic force microscopy (AFM) can be combined with fluorescence microscopy to measure the changes in intracellular calcium levels (indicated by fluorescence of Ca2+ sensitive dye fluo-4) in response to mechanical stimulation performed by AFM. Mechanical stimulation using AFM is associated with cantilever movement, which may interfere with the fluorescence signal. The motion of the AFM cantilever with respect to the sample resulted in changes of the reflection of light back to the sample and a subsequent variation in the fluorescence intensity, which was not related to changes in intracellular Ca2+ levels. When global Ca2+ responses to a single stimulation were assessed, the interference of reflected light with the fluorescent signal was minimal. However, in experiments where local repetitive stimulations were performed, reflection artifacts, correlated with cantilever motion, represented a significant component of the fluorescent signal. We developed a protocol to correct the fluorescence traces for reflection artifacts, as well as photobleaching. An added benefit of our method is that the cantilever reflection in the fluorescence recordings can be used for precise temporal correlation of the AFM and fluorescence measurements.

Keywords

AFMfluorescence artifactsmechanical stimulationcalcium signalingfluo-4
Type
Biological Applications: Techniques, Software, and Equipment Development
Information
Microscopy and Microanalysis , Volume 18 , Issue 4 , August 2012 , pp. 808 - 815
Copyright
Copyright © Microscopy Society of America 2012

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