OptoRheo: Simultaneous in situ micro-mechanical sensing and imaging of live 3D biological systems

Abstract

Biomechanical cues from the extracellular matrix (ECM) are essential for directing many cellular processes, from normal development and repair, to disease progression. To better understand cell-matrix interactions, we have developed a new instrument named ‘OptoRheo’ that combines light sheet fluorescence microscopy with particle tracking microrheology. OptoRheo lets us image cells in 3D as they proliferate over several days while simultaneously sensing the mechanical properties of the surrounding extracellular and pericellular matrix at a sub-cellular length scale. OptoRheo can be used in two operational modalities (with and without an optical trap) to extend the dynamic range of microrheology measurements. We corroborated this by characterising the ECM surrounding live breast cancer cells in two distinct culture systems, cell clusters in 3D hydrogels and spheroids in suspension culture. This cutting-edge instrument will transform the exploration of drug transport through complex cell culture matrices and optimise the design of the next-generation of disease models.

Fully angularly resolved 3D microrheology with optical tweezers

“…Therefore, by determining the gradient of the MSD for the first two lag-times (i.e., and , the shortest time delays is calculated for), one should derive a good estimation of , as recently corroborated by this group while measuring the apparent viscosity experienced by a bead approaching a hard interface in 3D (Mendonca, et al 2023 ). However, it must be noted that, in practice, the MSD values for the earliest lag times may be erroneously high if the real displacement is less than the resolution of the tracking system.…”

“…The optical trap was generated using a 1064-nm DPSS laser (Opus, Laser Quantum) with a maximum output of 5W on an inverted microscope set up (Olympus IX-73) with a high NA objective lens (LUMFLN60XW 60 × 1.1 NA 1.5 mm WD; Olympus). For tracking in 3D, a multiplane imaging system utilising a pair of gratings to produce nine images spatially separated on the image sensor each with a different focal plane was employed, the details of which are described elsewhere (Matheson et al 2021a , Matheson, et al 2021b ; Mendonca, et al 2023 ). In brief, a 4f image relay system consisting of two 300 mm lenses was set up in the detection path between the camera (Hamamatsu ORCA Flash 4.0 V2) and the microscope body, and the multiplane grating pair was placed in the telecentric position.…”

“…This theoretically offers the possibility of probing the viscoelastic properties of the sample in all directions simultaneously. This is particularly appealing for biological samples where viscoelastic properties will vary spatially in all three dimensions, and could be highly anisotropic at a given point (Hasnain and Donald 2006 ; Mendonca, et al 2023 ). Furthermore, it is well established that in the proximity to an interface, the viscosity as obtained from microrheology may vary significantly with angle (Leach, et al 2009 ; Schäffer, et al 2007 ), providing an additional possible application for full angular resolution.…”

“…In recent work from this group, it has been shown that using the standard 2D analytical tools on a trap with a degree of anisotropy of 30% or higher can result in a substantial underestimation of the trap strength, and an overestimation of the fluid viscosity in certain directions (Matheson et al 2021a ). Crucially, as a consequence of diffraction and the focal spot being stretched along the axis of laser beam propagation, the difference in trap strength between versus and has been observed to exceed this level of anisotropy (Matheson et al 2021a , Matheson, et al 2021b ; Mendonca, et al 2023 ). This is without even considering other aberrations in the optical system may also introduce additional sources of trap anisotropy (Bowman, et al 2010 ; Dutra, et al 2007 , 2012 ; López-Quesada, et al 2009 ; Roichman, et al 2006 ).…”

Atomic Force Microscopy of Phytosterol Based Edible Oleogels

“…This may be passive, where beads are observed freely undergoing thermal motion, or active microrheology, where an optical trap is used to constrain the bead. The spatial resolution of this technique is typically on the scale of microns, much larger than the nano-scale resolution probed by AFM, but it does allow for a full 3D viscosity map to be developed and may provide information on the anisotropic viscoelastic response of the material in different directions [ 36 ].…”