Assistant Professor Wayne State University Detroit, Michigan, United States
Introduction:: Confocal Brillouin microscopy can quantify the mechanical properties of materials with sub-micrometer resolution and in a noncontact, nonperturbative, and label-free manner. It has been demonstrated as an emerging complementary tool to conventional methods in biophysics, bioengineering, and mechanobiology. Despite the advancement, one challenge encountered by confocal Brillouin microscopy is its slow acquisition speed (20-200 ms), which is inherently limited by the extremely low scattering efficiency of the spontaneous scattering process. One approach to break this limit is to utilize the stimulated Brillouin scattering, which is a nonlinear process and can generate much stronger Brillouin signal. The stimulated Brillouin microscopy was developed by using two tunable continuous wave lasers, and mechanical imaging of C. elegans has been acquired with the dwell time as short as 20 ms. However, since the excitation of the stimulated process requires the presence of high laser intensity, the potential phototoxicity to various biological samples is still unclear and needs further assessment. In addition, both spontaneous and stimulated Brillouin microscopy conduct 3D mechanical mapping using point-scanning mode, where the laser beam is focused into a spot and each pixel is scanned by the laser spot in sequence. As such, the scanning process will introduce redundant light dose to the out-of-focus pixels along the beam propagation path. Together, the slow acquisition speed and high light dose bring about challenges for volumetric mapping of biological samples using current Brillouin technique, and the challenge becomes more severe as the sample size is larger.
Materials and Methods:: Here, we introduce a dual line-scanning Brillouin microscopy (dLSBM) that improves the acquisition speed by one order of magnitude and significantly reduces the light dose of optical illumination for 3D mapping of biological samples (Fig.1a). The idea of dLSBM originated from one fact of the spontaneous Brillouin scattering process with confocal configuration, where the most majority of the light does not contribute to Brillouin signal but propagates away from the focal plane. The reuse of this non-scattered light could generate extra Brillouin signal and improve overall efficiency. To this end, we proposed a multiplexing configuration that shares the same principle of light-sheet microscopy: by placing the illumination path and the detection path in a 90-degree configuration, Brillouin signals of multiple points along the beam line can be collected simultaneously by reusing the light of the illumination beam (Fig. 1b). The dLSBM works in the regime of spontaneous scattering process, thus relatively low laser intensity is needed. More importantly, since all the points on the illumination path are measured with one shot, there is no redundant light dose introduced to the sample during volumetric mapping.
Results, Conclusions, and Discussions:: We first validated the performance of the dLSBM by comparing it to a confocal Brillouin microscope with similar but slightly better resolution (NA=0.4). By measuring the same spheroid, detailed structure acquired by dLSBM is similar to the confocal Brillouin microscope (Fig.1d&1e). The histogram distributions of the scanned sections indicate dLSBM has successfully captured the same mechanical features of the spheroid as the confocal Brillouin microscope.
Next, we collected multiple cross-sectional Brillouin images of a spheroid by rotating the sample within the illumination-detection plane for 3D imaging reconstruction, from where we can clearly observe the spatial heterogeneity of biomechanics within a multicellular system (Fig.1c). We also tested the cell viability after 3D mapping and confirmed that there was no photodamage under current light dose (Fig.1g&1h). For N=200 in our setup, the light dose introduced by the dLSBM is only 7.4% of that introduced by confocal Brillouin microscopy (Fig.1f).
The improved acquisition speed of dLSBM allows us to monitor the quick mechanical response of the spheroid to external stimulus. To demonstrate this, we tracked the Brillouin shift of the spheroids in real time as they were exposed to hyperosmotic and hypoosmotic shocks. We observed the spheroids behaved similarly as single cells under different osmotic conditions: the Brillouin shift of the spheroids increased under hyperosmotic shock and decreased under hypoosmotic shock, along with the volume change in the opposite direction. The correlation between cell volume and cell stiffness has been elucidated on single cell level, but much less is understood in spheroid. Since the cell volume and stiffness change relates to many functions and behaviors including cell proliferation, migration, differentiation, and cancer progression, our instrument provides a sensitive tool to study cell dynamics in the multicellular system.
Acknowledgements (Optional): :
References (Optional): : Zhang, J., Nikolic, M., Tanner, K. et al. Rapid biomechanical imaging at low irradiation level via dual line-scanning Brillouin microscopy. Nat Methods (2023). https://doi.org/10.1038/s41592-023-01816-z