Optical studies of the dynamics and mechanics inside live biological cells: from molecular to collective phenomena
Presented by Kareem Elsayad, PhD, Vienna Biocenter Core Facilities (VBCF), Vienna Biocenter, Austria
I will talk about several optical techniques we have developed and are developing to study individual and collective molecular dynamics (mechanical properties), and how these are giving us unique insight into the underlying physics, chemistry and function of biological cells, as well as in some cases their potential for diagnostic applications.
Firstly, I will discuss a form of label-free optical spectroscopy -Brillouin light scattering microscopy- we have employed to map the acoustic phonon velocity, and thereby also the complex longitudinal elastic modulus in 3D within live cells. Using a custom imaging-spectrometer we have for the first time demonstrated non-invasive studies in live tissue and the possibility to correlate mechanical changes to molecular composition and structural features with confocal near-diffraction limited optical resolution. Doing so we are able to map changes in the elastic moduli and effective viscosity in diverse live biological samples spanning developing embryos to stem cells to the extracellular matrix of plant cells in response to different chemical/environmental conditions and underlying structural changes. Using a variation of this setup we can also measure the in-plane wavevector dependence of the acoustic phonon velocity in a single shot and spatially map this in 3D in live cells. This gives us access to all the diagonal compressive components of the stiffness tensor and thereby the mechanical anisotropy throughout a sample. These are not only essential for accurate modelling, but have the potential to be prognostic indicators (via the fractional anisotropy) of diverse pathologies. For plant cell walls we are thereby able to quantitatively predict the relative differences in the anisotropic elastic moduli (and thereby indirectly the cell shape) as confirmed using super-resolution microscopy measurements of the microfibril orientation and effective medium theories. I will also discuss a novel total-internal reflection implementation of Brillouin microscopy which allows us to couple to the transverse acoustic phonon modes and thus measure the shear wave velocity and complex shear modulus, which we are currently applying to study dynamic phase transitions in lipid-bilayers.
Secondly, I will talk about an approach we have developed for the measurement of fast molecular diffusion coefficients employing a streak camera – which can give us insight into the molecular dynamics underlying viscoelastic properties. Using a trick of superimposing temporal projections at different intervals we are able to access fast diffusion coefficients (including rotational diffusion) over an entire line in a fraction of the time required for conventional point- Fluorescence Correlation Spectroscopy (FCS), and at correlation times far beyond that accessible by imaging-FCS. This on the one hand can serve as a useful tool for parallelized FCS, but also gives us insight into the distribution of mechanical relaxation times and interpretation of the high-frequency regime measured using Brillouin scattering in live cells. Importantly the latter has the potential to directly connect the mechanical properties inferred from molecular dynamics (via correlation spectroscopy) to the those derived from collective phenomena (via Brillouin spectroscopy).
Finally, I will discuss a microscopy method we have developed for improving the axial resolution down to 5-10nm in the vicinity of metal-dielectric coated substrates that is also suitable for live cell studies. The effect makes use of the interference of light scattered from the substrate, together with subtle change in the measured emission spectra of fluorophores (due to interference with light reflected from the substrate), and has allowed us to study the dynamics of cell adhesion proteins and filopodia with deep sub-diffraction limit optical resolution.
This is a Zoom event -- link will be posted before the event
11:00 am, Wednesday, March 25, 2020
No Room Specified, Physics & Astronomy
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