Ultrasound-induced reorientation for multi-angle optical coherence tomography.

Organoid and spheroid technology provide valuable insights into developmental biology and oncology. Optical coherence tomography (OCT) is a label-free technique that has emerged as an excellent tool for monitoring the structure and function of these samples. However, mature organoids are often too opaque for OCT. Access to multi-angle views is highly desirable to overcome this limitation, preferably with non-contact sample handling. To fulfil these requirements, we present an ultrasound-induced reorientation method for multi-angle-OCT, which employs a 3D-printed acoustic trap inserted into an OCT imaging system, to levitate and reorient zebrafish larvae and tumor spheroids in a controlled and reproducible manner. A model-based algorithm was developed for the physically consistent fusion of multi-angle data from a priori unknown angles. We demonstrate enhanced penetration depth in the joint 3D-recovery of reflectivity, attenuation, refractive index, and position registration for zebrafish larvae, creating an enabling tool for future applications in volumetric imaging.

Controlled orientation and sustained rotation of biological samples in a sono-optical microfluidic device.

In cell biology, recently developed technologies for studying suspended cell clusters, such as organoids or cancer spheroids, hold great promise relative to traditional 2D cell cultures. There is, however, growing awareness that sample confinement, such as fixation on a surface or embedding in a gel, has substantial impact on cell clusters. This creates a need for contact-less tools for 3D manipulation and inspection. This work addresses this demand by presenting a reconfigurable, hybrid sono-optical system for contact-free 3D manipulation and imaging, which is suitable for biological samples up to a few hundreds of micrometers in liquid suspension. In our sono-optical device, three independently addressable MHz transducers, an optically transparent top-transducer for levitation and two side-transducers, provide ultrasound excitation from three orthogonal directions. Steerable holographic optical tweezers give us an additional means of manipulation of the acoustically trapped specimen with high spatial resolution. We demonstrate how to control the reorientation or the spinning of complex samples, for instance for 3D visual inspection or for volumetric reconstruction. Whether continuous rotation or transient reorientation takes place depends on the strength of the acoustic radiation torque, arising from pressure gradients, compared to the acoustic viscous torque, arising from the shear forces at the viscous boundary layer around the particle. Based on numerical simulations and experimental insights, we develop a strategy to achieve a desired alignment or continuous rotation around a chosen axis, by tuning the relative strengths of the transducers and thus adjusting the relative contributions of viscous and radiation torques. The approach is widely applicable, as we discuss in several generic examples, with limitations dictated by size and shape asymmetry of the samples.

Cortical contractility triggers a stochastic switch to fast amoeboid cell motility.

3D amoeboid cell migration is central to many developmental and disease-related processes such as cancer metastasis. Here, we identify a unique prototypic amoeboid cell migration mode in early zebrafish embryos, termed stable-bleb migration. Stable-bleb cells display an invariant polarized balloon-like shape with exceptional migration speed and persistence. Progenitor cells can be reversibly transformed into stable-bleb cells irrespective of their primary fate and motile characteristics by increasing myosin II activity through biochemical or mechanical stimuli. Using a combination of theory and experiments, we show that, in stable-bleb cells, cortical contractility fluctuations trigger a stochastic switch into amoeboid motility, and a positive feedback between cortical flows and gradients in contractility maintains stable-bleb cell polarization. We further show that rearward cortical flows drive stable-bleb cell migration in various adhesive and non-adhesive environments, unraveling a highly versatile amoeboid migration phenotype.

SLM-based off-axis Fourier filtering in microscopy with white light illumination.

In various microscopy applications spatial light modulators (SLMs) are used as programmable Fourier filters to realize different optical contrast enhancement methods. It is often advantageous to use the SLM in off-axis configuration, where the filtered image wave is sent into the first diffraction order of a blazed grating superposed to the phase mask on the SLM. Because of dispersion this approach is, however, typically limited to spectrally narrowband illumination. Here we suggest a method involving a grating for pre-compensation, which allows one to use spectrally broadband (even thermal) light in SLM-based Fourier filtering. The proposed approach is demonstrated by multicolor imaging of amplitude and phase objects, such as a resolution target, onion epidermal cells and human epithelial cheek cells.

Quantitative analysis of shape and volume changes in activated thrombocytes in real time by single-shot spatial light modulator-based differential interference contrast imaging.

We suggest to use a combination of optical tweezers and single-image quantitative differential interference contrast (DIC) emulated by a spatial light modulator (SLM) to study physiological shape changes in thrombocytes after activation and demonstrate the effectiveness of this system for the given task. A specially designed phase mask displayed at the SLM enables quantitative phase calculation from only a single recording. The optical tweezers stabilize trapped thrombocytes for long-time monitoring of changes in the optical thickness profile of thrombocytes during activation by adenosine diphosphate (ADP).

In vitro investigation on the pH dependence of the absorption and fluorescence properties of the photosensitizer mTHPC.

Fluorescence excitation efficiency is of great importance for photodynamic diagnosis. Because usually a difference in the interstitial pH between normal and tumor tissue occurs, it is necessary to assess the impact of pH on the fluorescence emission intensity of the photosensitizer meta-tetrahydroxyphenylchlorin (mTHPC) in this context. The results obtained by in vitro fluorescence measurements clearly indicate that pH values below 6 lead to a significant decrease in the fluorescence intensity. In the physiological range of pH 6.5-7.2, however, no pH dependence was found. Besides the decrease in the fluorescence intensity of mTHPC for pH < 6, changes in the spectral shape of the absorption were found. These changes can be utilized for "dual-wavelength ratio imaging," using mTHPC as a pH-sensitive indicator with the excitation pair 405 nm/436 nm in the range of pH 3.5-6.