Quantitative optical microscopy for aberrating and scattering biological media
Biophysics
Optics, Quantum Optics and Physics of Atoms, Molecules and Plasmas
Statistical Physics, Nonlinear Dynamics, Complex Systems, Soft and Fluid Matter, Biological Physics
Final Report Abstract
Optical microscopy in biology develops today along two major directions: improving imaging quality, especially in terms of resolution and 3D field of view; and improving content and precision of associated measurements, such as determining molecular concentration and dynamics. For obtaining the latter information, a very successful and well-established technique is Fluorescence Fluctuation Microscopy (FFM), which is a generalisation of classical Fluorescence Correlation Spectroscopy (FCS) that exploits different microscopy techniques. Our project aimed at better understanding how light scattering and optical aberrations disturb FFM measurements, and how to develop schemes for correcting these effects. The project was divided into two major parts: Experimental measurements that should give a better understanding of fluorescence excitation and detection through scattering media, and exact theoretical modelling, of light propagation through scattering objects. Moreover, we worked out a full theoretical framework of optimal image correction for a given microscopy hardware with arbitrary aberrations. On the experimental side, we performed fluorescence correlation spectroscopy (FCS) measurements through dense layers of scattering particles. These particles were polymer beads of known diameter, which were arranged in randomly structured layers that served as a scattering medium of well-defined scattering characteristics. We studied how the results of FCS measurements (molecular brightness, diffusion time, detection volume) were changed as a function of relative position between laser focus and scattering layer. We found that already at moderate distances between scattering layer and detection volume (focus position), the FCS results approached values close to those obtained in absence of any scattering material. This is an important information, because it shows that scattering of excitation and fluorescence light on randomly distributed scatterers affects quantitative results of FCS much less than expected. Moreover, we could show that the speckles generated by scattering of the excitation light can be used directly for performing wide-field correlation spectroscopy through a scattering substrate, with no further corrections. On the theoretical side, we developed an exact vector wave-optical model for the focusing of a Gaussian laser beam through a dielectric bead and for the detection of fluorescence through this bead, and we used this model for calculating exact FCS curves. We then mapped the theoretically estimated diffusion time and detection volume as a function of the focus position with respect to the bead. These calculations are the fundament for further modelling of how a layer of many scattering beads distorts/affects an FCS measurement. However, already the single-bead calculations confirm the experimental results that the impact of wavefront-distorting beads on FCS measurements diminishes very rapidly already at moderate distances between focus and bead. Finally, within the context of understanding the impact of aberrations on imaging quality (and thus also FCS quality) of an optical microscope, we developed a general theory of perfect imaging systems and found an exact description of the most optimal Optical Transfer Function (OTF) of optical microscopes, which is valid for any bandwidth-limited imaging instrument in general.
Publications
- "Confocal fluorescence correlation spectroscopy through a sparse layer of scattering objects" Optics Express 27(14) (2019): 19382-19397
Sarkar, Anirban, Joseph Gallagher, Irène Wang, Giovanni Cappello, Jörg Enderlein, Antoine Delon, and Jacques Derouard
(See online at https://doi.org/10.1364/oe.27.019382) - "Quantitative analysis of hidden particles diffusing behind a scattering layer using speckle correlation" Optics Express 28(22) (2020): 32936-32954
Sarkar, Anirban, Irène Wang, Jörg Enderlein, Jacques Derouard, and Antoine Delon
(See online at https://doi.org/10.1364/oe.401506)
