Confocal Brillouin microscopy for three-dimensional mechanical imaging

Giuliano Scarcelli, Seok Hyun Yun
2007 Nature Photonics  
Acoustically induced inelastic light scattering, first reported in 1922 by Brillouin 1 , allows non-contact, direct readout of the viscoelastic properties of a material and has widely been investigated for material characterization 2 , structural monitoring 3 and environmental sensing 4 . Extending the Brillouin technique from point sampling spectroscopy to imaging modality 5 would open up new possibilities for mechanical imaging, but has been challenging because rapid spectrum acquisition is
more » ... quired. Here, we demonstrate a confocal Brillouin microscope based on a fully parallel spectrometer-a virtually imaged phased array-that improves the detection efficiency by nearly 100-fold over previous approaches. Using the system, we show the first cross-sectional Brillouin imaging based on elastic properties as the contrast mechanism and monitor fast dynamic changes in elastic modulus during polymer crosslinking. Furthermore, we report the first in situ biomechanical measurement of the crystalline lens in a mouse eye. These results suggest multiple applications of Brillouin microscopy in biomedical and biomaterial science. The mechanical properties of biological tissues and biomaterials are closely related to their functional abilities 6 , and thus play significant roles in many areas of medicine. For example, coronary arteries hardened by calcification can cause heart problems, mechanically weakened bones resulting from osteoporosis represent a serious orthopaedic concern, and the stiffness of the extra-cellular matrix influences drug delivery and cell motility 7 . As such, the ability to measure mechanical properties non-invasively in vivo at the microscopic (cellular) scale would have a wide range of biomedical applications, as well as uses in material science and engineering 8, 9 . Conventional mechanical tests, such as dynamic mechanical analysis and rheometry, require mechanical forces to be applied to samples through contact and, although accurate and comprehensive, they are therefore not well suited to in situ highresolution measurements. Miniaturized mechanical methods have become increasingly sophisticated 10 , but they are still limited to surface measurement. Elastography is a promising technique used to extract mechanical information from structural deformation images 11 ; however, sensitivity to the assumed stress distribution and physiological motion limit spatial resolution and measurement precision. Acoustic techniques, such as ultrasound and acoustic microscopy, are non-invasive and can offer microscopic resolution. However, these techniques tend to be effective only for samples with simple internal structures 12 , because the detected signals (acoustic echoes) originate from the spatial derivative of the acoustic impedance to be measured, rather than its absolute magnitude. Spontaneous Brillouin scattering is an inelastic scattering process arising from inherent density fluctuations, or acoustic phonons, in the medium (Fig. 1a) . Brillouin spectroscopy measures spectral changes upon scattering, providing direct information on the phonon's properties that are closely related to the viscoelastic properties of the medium (see Methods). Previously, Brillouin spectroscopy has been used successfully to measure the viscoelastic properties of samples ex vivo, including collagen fibres 13,14 , bone 15 , cornea and crystalline lens 16, 17 . Nevertheless, this technique has remained a point sampling method, mainly because the measurement time to perform imaging was too long, typically minutes per spectrum (pixel). A challenge in Brillouin spectroscopy is separating Brillouin light from elastically scattered light, which arises from Rayleigh and Mie scattering or reflections from optical components. This can be orders of magnitude stronger than Brillouin scattered light in most biological tissues. In addition, the Brillouin frequency shift, in the order of GHz (0.1-0.5 cm 21 ), is too small to resolve with conventional spectrometers. For high spectral resolution and extinction, Brillouin spectroscopy has relied on multiple-pass scanning Fabry-Perot (FP) interferometers 18 , grating-based monochromators 19 and optical beating methods 20 . These scanning approaches are slow, however, as they measure individual spectral components sequentially. A non-scanning parallel approach has been demonstrated recently using an angle-dispersive FP etalon 5,21 . However, at each beam incidence angle, only a specific narrowband spectral component is transmitted and detected, while the rest is reflected. Consequently, in both the non-scanning and the scanning approaches, the throughput efficiency is fundamentally limited to less than 1/F, where F denotes the finesse of etalon; this results in a trade-off between measurement resolution and acquisition speed. To circumvent this problem, we developed a fully parallel dispersive imaging spectrometer based on a virtually imaged phased array 22,23 (VIPA) (Fig. 1b; see also Methods). Our custombuilt VIPA spectrometer featured high finesse of up to 56 and high throughput efficiency over 80%, and has a free spectral range (FSR) of 33.3 GHz. The spectrometer was integrated into a homebuilt confocal microscope set-up (Fig. 1c) . Using a single-mode fibre ensured strict confocality, flexible beam delivery, and minimal stray light in the spectrometer. We used a dual-axis configuration 24 LETTERS nature photonics | VOL 2 | JANUARY 2008 | www.nature.com/naturephotonics 39
doi:10.1038/nphoton.2007.250 pmid:19812712 pmcid:PMC2757783 fatcat:csuflaczbfcgdjgypjr5zvbkky