Abstract
Second harmonic generation (SHG) microscopy is a powerful tool for label free ex vivo or in vivo imaging, widely used to investigate structure and organization of endogenous SHG emitting proteins such as myosin or collagen. Polarization resolved SHG microscopy renders supplementary information and is used to probe different molecular states. This development towards functional SHG microscopy is calling for new methods for high speed functional imaging of dynamic processes. In this work we present two approaches with linear polarized light and demonstrate high speed line scan measurements of the molecular dynamics of the motor protein myosin with a time resolution of 1 ms in mammalian muscle cells. Such a high speed functional SHG microscopy has high potential to deliver new insights into structural and temporal molecular dynamics under ex vivo or in vivo conditions.
Highlights
Since the discovery of second harmonic generation (SHG) signals generated in endogenous proteins like myosin in muscle [1,2,3,4,5,6] or collagen [2, 7] various advances in this field of microscopy have been established: The intrinsic nature of the SHG signal allows for label free and high contrast imaging under in vivo [8] or ex vivo [2] conditions, making SHG microscopy a powerful tool in biomedical research
Polarization resolved structural SHG (sSHG) microscopy was performed by measuring over a range of polarization angles of the incident laser beam, at different characteristic angles, by splitting the SHG signal itself in different polarizations or by using Stokes vector analysis [19,20,21,22,23,24] to gather information of structural order and orientation
We demonstrate high speed line scan measurements with millisecond time resolution during muscle contraction, constituting another important step to establish fast functional SHG microscopy in order to investigate molecular dynamics
Summary
Since the discovery of second harmonic generation (SHG) signals generated in endogenous proteins like myosin in muscle [1,2,3,4,5,6] or collagen [2, 7] various advances in this field of microscopy have been established: The intrinsic nature of the SHG signal allows for label free and high contrast imaging under in vivo [8] or ex vivo [2] conditions, making SHG microscopy a powerful tool in biomedical research. By demonstrating the changes in the polarization dependent SHG signal due to conformational changes of the motor protein myosin in different contraction states of muscle (rigor or relaxed state, isometric tetanic contraction) [6, 25] first steps towards a functional SHG (fSHG) have been realized. These results aim for a method that allows retrieving functional information on the molecular level during muscle contraction
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