Functional second harmonic generation microscopy probes molecular dynamics with high temporal resolution

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.

Monitoring dynamic collagen reorganization during skin stretching with fast polarization‐resolved second harmonic generation imaging

“…The mean ± SEM of every parameter is also calculated samples, as demonstrated in Figure 2. Moreover, it works in thick samples where epi-detection is mandatory, which is not the case for many other approaches [22][23][24][25]35]. It may be further improved by synchronizing the EOM with pixel imaging in order to image tissues with faster motion.…”

“…This acquisition time is only moderately longer than the one relevant for morphological filtering in one pixel, that is the acquisition time of the window used for filtering (in our case, 19 × 19 pixels): 5.4 ms. Some studies have proposed to further reduce the acquisition time by using only two or three polarization states either for the excitation or for the analysis of circularly excited SHG . While this approach may be efficient in samples with simple orientation distribution, it cannot provide accurate orientation maps in samples with more complex orientation distribution.…”

“…Reliable P‐SHG‐based determination of the collagen reorganization during dynamic processes is therefore impossible if the sample exhibits distortions on the time scale needed to perform measurements with different polarizations. A number of strategies have been developed to overcome this limitation and have proven efficient in specific samples . Nevertheless, most of these approaches are based on polarization analysis of forward SHG signals, which is not applicable to thick scattering tissues, or they use only two incident polarizations for SHG imaging, which limits their accuracy in tissues with disordered collagen.…”

Technological Challenges in the Development of Optogenetic Closed-Loop Therapy Approaches in Epilepsy and Related Network Disorders of the Brain

“…These tools allow the user to estimate the illumination profile and the total volume of activated neural tissue based on the properties of the optical source [ 108 ]. Previous papers reported results of simulations to demonstrate the effect of the numerical aperture (NA) and the light source [ 13 , 107 , 109 ]. The NA and the size of the source showed to have only a limited effect on the illumination profile and on the volume of activated tissue, as a result of the large scattering coefficient of brain tissue.…”

Dual‐ and single‐shot susceptibility ratio measurements with circular polarizations in second‐harmonic generation microscopy

“…Staggered laser pulses for fast polarization switching can also be used . Fast polarimetric detection is usually accomplished by measuring several polarization states in parallel on separate detectors . For example, a single‐shot (scan) orientation‐independent polarimetric measurement of the R‐ ratio was accomplished with circularly polarized incident light and three parallel detectors acquiring different linear SHG polarizations .…”