Simultaneous Advanced Microscopies for Live Cell Signaling Dynamics Investigations

Abstract

We present a new type of combined microscopy for the life sciences based on the integration of differential spinning disk fluorescence optical sectioning microscopy and nanomechanical mapping Atomic Force Microscopy (AFM).[1] Our hardware platform creates the opportunity for simultaneous acquisition of membrane receptor recognition maps and fluorescence optical sectioning images of live cells, providing new experimental capabilities in cell signaling investigations.Simultaneous spatio-temporal generation of AFM and fluorescence microscopy data is made possible through time-independent illumination at low light intensities of the AFM cantilever that strongly reduces the interaction of AFM cantilever motion with fluorescence excitation light as compared to existing hardware integration platforms based on confocal laser scanning microscopy and AFM. Namely, fluorescence excitation light has the capability to induced cantilever heating and subsequent sample heating through heat conduction. We identify several system specific noise-sources that can interfere with live cell investigations and describe solutions to mitigate them.[1] Miranda, A., Martins, M. & De Beule, P. A. A. Simultaneous differential spinning disk fluorescence optical sectioning microscopy and nanomechanical mapping atomic force microscopy. Rev. Sci. Instrum. 86, 093705 (2015).