Pulsed stimulated Brillouin microscopy enables high-sensitivity mechanical imaging of live and fragile biological specimens

Abstract

Brillouin microscopy is an emerging optical elastography technique capable of assessing mechanical properties of biological samples in a 3D, all-optical and hence non-contact fashion. The typically weak Brillouin scattering signal can be substantially enhanced via a stimulated photon-phonon process, which improves the signal-to-background ratio (SBR) as well as provides higher spectral resolution. However, current implementations of stimulated Brillouin spectroscopy (SBS) require high pump powers, which prohibit applications in many areas of biology, especially when studying photosensitive samples, or when live-imaging in 3D and/or over extended time periods. Here, we present a pulsed SBS scheme that takes full advantage of the non-linearity of the pump-probe interaction in SBS. In particular, we show that through quasi-pulsing and diligent optimization of signal detection parameters, the required pump laser power can be decreased ~20-fold without affecting the signal levels or spectral precision. Moreover, we devise a custom analysis approach that facilitates the analysis of complex, multi-peaked Brillouin spectra in order to harness the high spectral resolution of SBS for the specific identification of biomechanical components inside the point-spread function of the microscope. We then demonstrate the low-phototoxicity and high-specificity of our pulsed SBS approach by imaging sensitive single cells, zebrafish larvae, and mouse embryos as well as adultC. eleganswith sub-cellular detail. Furthermore, our method permits observing the mechanics of organoids andC. elegansembryos over time. We expect that the substantially lower photo-burden and improved SBR of pulsed SBS will facilitate studying biomechanics in 3D at high spatio-temporal resolution in living biological specimens in a non-invasive manner, opening up exciting new possibilities for the field of mechanobiology.

Measuring age-dependent viscoelastic properties of organelles, cells and organisms via Time-Shared Optical Tweezer Microrheology

“…8). Together, this indicates that endogenous lipid droplets are sufficiently stiffer than other organelles, as also can be seen in their higher longitudinal modulus[58, 59], and are suitable as nano-indenters for optical force measurements.…”

Brillouin gain microscopy

“…Despite the substantial improvements enabled by cw SBM for optical mechanical imaging, its temporal resolution is still limited to a few tens of milliseconds under excitation power of a few hundreds of milliwatts, requiring caution with the high irradiation dose when imaging biological samples. Recently, pulsed SBM has alleviated this need, although the contrast presented by either the peak SBG or the Brillouin linewidth appears to be low 22,23 .…”

Non-contact and label-free biomechanical imaging: Stimulated Brillouin microscopy and beyond

“…For example, a very recent work shows ten times improvement in the acquisition speed can be achieved by optimizing the detection using localization theory [ 40 ]. Furthermore, the efficiency of SBM can be improved by replacing cw operation with quasi-pulsed operation [ 44 ]. By optimizing the pump-probe interaction at high peak power and short interaction time, the quasi-pulsed SBM can reduce the illumination power by 20 times while maintaining the signal quality.…”

“…To date, the feasibility of cw and quasi-pulsed SBMs has been demonstrated in liquid (e.g., water, methanol, intralipid solution) [ 43 , 45 ], C. elegans [ 39 ], single cell, and organoids with microscopic resolution [ 44 ], suggesting promising potential of SBM as a new biomechanical imaging modality. Importantly, in addition to the Brillouin shift and linewidth, cw SBM can probe the density of the material.…”