The nucleus is the largest and probably stiffest organelle of eukaryotic cells. As such, its mechanical properties are tightly related to various cell functions. However, the mechanical behavior of the nucleus within intact cells is much less know because it is embedded in the cytoplasm thus not directly accessible by the existing contact-based technique. In this work, the regulation of nuclear mechanics was investigated by all-optical Brillouin microscopy and the results were found to be consistent with three-dimensional chemomechanical modeling. We found the intact nuclear mechanics is regulated by both the cytoskeletal networks and internal nanostructure.
KEYWORDS: Live cell imaging, Microscopy, Light scattering, Laser scattering, Environmental sensing, Signal to noise ratio, Scattering, Solid state lasers, Solid state physics, Microscopes
In Brillouin microscopy, absorption-induced photodamage of incident light is the primary limitation on signal-to-noise ratio in many practical scenarios. 660 nm may represent an optimal wavelength for Brillouin microscopy as it offers minimal absorption-mediated photodamage at high Brillouin scattering efficiency. We demonstrate that live cells are ~80 times less susceptible to the 660 nm incident light compared to 532 nm light, which overall allows Brillouin imaging with more than 30 times higher signal intensity. We apply this improved Brillouin microscope to analyze the response of human glioblastoma cells to a range of in vitro biomimetic environments.
The nucleus is the largest and stiffest organelle of eukaryotic cells, and as such, its mechanical properties are tightly related to various cell functions. Many efforts have been devoted to characterize the mechanical properties of nucleus, but the current techniques generally need physical contact of the cell and staining of the nucleus and thus cannot acquire the mechanical information directly. Brillouin microscope is an integration of a confocal microscope and a Brillouin spectrometer, which measures the spectral shift due to the spontaneous Brillouin light scattering, and from that the longitudinal modulus of the sample can be quantified. In this work, by combining the standard Brillouin microscope with the microfluidic technique, we developed a Brillouin flow cytometry that can quantify the mechanical properties of the intact cellular nucleus in a non-contact and label-free manner. As cell flows through a microfluidic channel, its mechanical property at different regions will be sampled by a sub-micron beam spot of the Brillouin microscope. The mechanical information of the nucleus from the cell population can then be identified and extracted via data post-processing, which is further confirmed by co-registering Brillouin data with fluorescence data from the same cell. Currently, the overall throughput of this technique is about 200 cells per hour, mainly relies on the acquisition speed of the spectrometer, which could be readily improved with available technology. We verified the capability of this all-optical technique by distinguishing the stiffness changes of the nucleus that are relevant to physiological and pathological phenomena.
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