High-resolution line-scanning Brillouin microscopy for fast and low phototoxicity live-imaging of mechanical properties in biology

Abstract

Brillouin microscopy is a fully-optical technique that relies on the interaction between spontaneous, thermal generated soundwaves (phonons) and monochromatic laser light. Mechanical properties of biological samples can be inferred in 3D from the spectrum of the scattered light. A state-of-the-art Brillouin microscope consists of a confocal microscope coupled with a spectrometer that features an acquisition time ~<100ms. That is few orders of magnitude higher than a fluorescence confocal microscope, making it challenging to image fast process and/or photosensitive samples. To overcome this limitation, we implemented a line-scanning approach, that allows to acquire multiple spectra in a single camera acquisition, effectively reducing the acquisition time per point by <100x. We designed the system to achieve high resolution (0.8NA) within a FOV of 200μm and use near-IR wavelength for reduced photodamage. The illumination and detection objectives are mounted in a 90 degrees inverted ‘V’ configuration, that facilitates mounting and live-imaging of small organisms (e.g. mouse embryos) in physiological conditions. We furthermore implemented a concurrent fluorescence light-sheet imaging modality into our setup to correlate the mechanical signals to their underlying molecular constituents. Moreover, we developed a GPU-enhanced, fast numerical fitting routine appropriate for the orthogonal scattering geometry and to visualize the measured spectral properties in real-time and with high localization precision. To demonstrate the capabilities of our new microscope, we imaged mouse embryo development over 48h, from 16-cells stage to late blastocyst, without observing any photodamage.