Direct Three-Dimensional Measurement of Refractive Index via Dual Photon-Phonon Scattering

Abstract

We developed a microscopy technique that can measure the local refractive index without sampling the optical phase delay of the electromagnetic radiation. To do this, we designed and experimentally demonstrated a setup with two co-localized Brillouin scattering interactions that couple to a common acoustic phonon axis; in this scenario, the ratio of Brillouin frequency shifts depends on the refractive index, but not on any other mechanical and/or optical properties of the sample. Integrating the spectral measurement within a confocal microscope, the refractive index is mapped at micron-scale three-dimensional resolution. As the refractive index is probed in epi-detection and without assumptions on the geometrical dimensions of the sample, this method may prove useful to characterize biological cells and tissues.When light propagates inside a material, the phase of the electromagnetic wave is sensitive to the optical path, which fundamentally couples the geometrical path and the local index of refraction. Thus, methods to map the refractive index of a material (e.g. phase contrast microscopy [1,2], digital holography microscopy [3,4], optical coherence tomography [5,6], etc. [7-10]) are intrinsically indirect as they rely on the knowledge, assumption, or measurement of the spatial dimensions of the sample. Even when optical path delay and thickness of the sample are well characterized, we can only obtain the average refractive index along the beam propagation axis. This fundamental issue has important practical ramifications as it prevents performing spatiallyresolved measurements of the index of refraction. Mapping the distribution and variations of the local refractive index is potentially crucial to analyze mass density behavior in cell biology [11][12][13][14], cancer pathogenesis [13,15,16], and corneal or lens refraction [17][18][19].Several techniques in the past years have emerged to circumvent the coupling of geometrical path and refractive index in 3D samples using a tomographic approach, i.e. performing multiple measurements from different angles to reconstruct the internal refractive index distribution [20][21][22][23][24]. Tomographic phase microscopy enabled spatiallyresolved measurements of refractive index for the first time. However, as the individual measurements are still based on optical path delay, known geometrical boundary conditions and/or reference refractive index values are needed as well as access to the sample from at least two sides. In addition, even under these conditions, the measurements are subject to artifacts due to phase wrapping when phase variations inside the sample are not smooth [1,2,13].Here we present a novel microscopy technique that probes the refractive index of materials relying on photon-phonon interactions, not optical path delays, and thus decouples optical from geometrical path. The technology is based on measuring two inelastic Brillouin scattering interactions in confocal configuration, probing the same acoustic phonon axis so that the refractive index is the ...

Correlative all-optical quantification of mass density and mechanics of subcellular compartments with fluorescence specificity

“…Only recently, serial Brillouin measurements of samples illuminated under different illumination angles allowed measuring the RI value inside the focal volume as well (Fiore, 2019). However, this technique requires illuminating the sample from two different directions, which doubles the acquisition time and decreases the spatial resolution of the measurement when compared to a setup only acquiring the Brillouin shift.…”

Optical elastography and tissue biomechanics

“…11 Thus Dr. Scarcelli's group advocated for high-precision refractive index measurements and discussed a newly developed confocal microscopy technique that can map the refractive index of samples in an absolute manner, with three-dimensional (3-D) resolution. 12 The unique experimental arrangement permits the same optical axis to be probed with two scattering geometries for Brillouin and elastic scattering, within a confocal voxel. In the proof-of-principle experiments, Dr. Scarcelli's group has achieved a refractive index accuracy of 0.001 and micron scale spatial resolution.…”

Recent progress and current opinions in Brillouin microscopy for life science applications

“…A combined BM-ODT setup can extract longitudinal modulus by correlative analysis of the Brillouin frequency shift and RI distribution in the sample. Alternatively, two co-localized Brillouin scattering measurements at different scattering angles can also be used to decouple the local RI distribution from the Brillouin shift within the same probed confocal volume, albeit at currently lower spatial resolution (Fiore et al 2019).…”