Seeing Stiffness of Tissue with Light

August 4, 2016

How can you determine how stiff a piece of material, say, biological tissue, is? You may want to poke it, then check how much its shape is altered; or you may even want to cut off a small piece, then torture it by stretching, squeezing, or pressing, and then measure how much you can deform it.

Figure 1. Principle of Brillouin scattering

Figure 1. Principle of Brillouin scattering

Conventionally, in biomedicine, when a destructive mechanical test is not involved, localized biomechanical properties are quantified with ultrasonic waves, which in nature is a mechanical wave. Recently, a light-based method, Brillouin scattering imaging, has been drawing attention due to its unique advantages in measuring mechanical properties in a non-contact, non-invasive fashion.

Brillouin scattering of light is one kind of inelastic scattering. It can be envisioned as the interaction between incoming photons and ultrasonic waves caused by intrinsic thermal dynamics. In principle, it looks very similar to Raman scattering – there is also a color-shift in the collected light that is scattered, but on a much smaller scale compared with Raman scattering, as is shown in Figure 1. This is similar to the Doppler effect in ultrasound.

Despite the fact that Brillouin scattering is approaching its centennial anniversary, and the first report of its application to biomedical research was published nearly 4 decades ago, this technique has not been widely used in the biomedical field due to its technical challenges.

First, the signal to detect in Brillouin scattering in soft tissue is rather small. The signals of spontaneous Brillouin scattering are orders of magnitude lower than elastic scattering, which mainly refers to Mie and Rayleigh scattering, as well as Fresnel reflection. Second, the color (wavelength) shift in Brillouin scattering is rather tiny – it is < 0.001 nm (this value is usually on the order of nm or tens of nm in Raman scattering). These challenges require two conditions for Brillouin imaging: a monochromatic source with narrow line-width, and a highly dispersive spectrometer to resolve the wavelength shift. Today these challenges are being overcome by engineers by better laser systems and high-efficiency spectrometers [1].

Thanks to success in these efforts, applications of newer versions of Brillouin imaging instruments in biomedicine have been explored in the last decade. The first imaging effort with a Brillouin spectrometer was carried out in 2005. A 2D map of a solid-liquid interface with 20 um spatial resolution was generated by Koski and Yarger [2]. The first in vivo imaging effort was reported by a team at Harvard Medical School, aiming at evaluating biomechanical properties of cornea and crystalline lens in animal models, and then in human in 2012. The authors confirmed age-related stiffness change in crystalline lens in mice [3], and elasticity alteration in the human cornea due to a medical condition called keratoconus [4]. In their study, 2D images of human corneas were generated based on average Brillouin frequency shift along axial-scanning at different locations (representative data shown in Figure 2).

Figure 2. Representative 2D Brillouin images of a healthy cornea and a cornea (A) with advanced keratoconus (B). Top row shows sagittal curvature and thickness map obtained with a Pentacam imaging device.

Figure 2. Representative 2D Brillouin images of a healthy cornea and a cornea (A) with advanced keratoconus (B). Top row shows sagittal curvature and thickness map obtained with a Pentacam imaging device.

Corneas and crystalline lenses are the most accessible and transparent tissue in biological bodies. As mentioned earlier, optical scattering hinders applications of this technique. Scientists have been able to find ways to filter out contamination in the signal due to undesired noise with better engineering solutions, to push this technique towards imaging turbid biological tissue. For example, a group led by Dr. Ykovlev from Texas A&M University has been conducting studies to quantify the difference in stiffness between healthy and diseased tissue in muscle and skin [5].

Figure 3. Representative 3D Brillouin image reconstruction of an NIH 3T3 cell cultured in a collagen gel matrix and the related top view obtained with bright-field microscopy (N = 5). Scale bars, 10 µm.

Figure 3. Representative 3D Brillouin image reconstruction of an NIH 3T3 cell cultured in a collagen gel matrix and the related top view obtained with bright-field microscopy (N = 5). Scale bars, 10 µm.

Cell mechanics is an important field for both fundamental and clinical research. One example is that, based on experimental evidence, researchers believe that understanding cancer cell deformability and its interactions with the extracellular environments offers enormous potential for significant new developments in disease diagnostics, prophylactics, therapeutics, and drug efficacy assays. However, conventional approaches to measure these properties requires physical contact between the sample and the probe, and is therefore destructive. There is also a lack of satisfactory resolution. Efforts have been directed at Brillouin imaging at a sub-cellular level. Scarcelli G. et al. [6] pioneered 3D intracellular hydromechanical properties imaging. 3D Brillouin maps revealed mechanical changes due to cytoskeletal modulation and cell-volume regulation in 2015. Meng et al. [7] reported their work on probing viscoelastic properties of individual red blood cells. Brillouin microscopy opens up new doors for investigation of biomechanical properties of cells and their microenvironment at superior resolution.

In conclusion, Brillouin imaging is a powerful tool for quantifying mechanical properties in biomedicine. Its scalable resolution and non-invasive and non-contact nature have great potential for both clinical practice and fundamental research.

  1. G. Scarcelli and S. H. Yun, “Confocal Brillouin microscopy for three-dimensional mechanical imaging,” Nature Photonics 2, 39-43 (2007).
  2. J. L. Y. K. J. Koski, “Brillouin imaging,” Applied Physics Letters 87, 061903 (2005).
  3. G. Scarcelli, P. Kim, and S. H. Yun, “In Vivo measurement of age-related stiffening in the crystalline lens by brillouin optical microscopy,” Biophysical Journal 101, 1539-1584 (2011).
  4. G. Scarcelli, S. Besner, R. Pineda, P. Kalout, and S. H. Yun, “In vivo biomechanical mapping of normal and keratoconus corneas,” JAMA Ophthalmology 133, 480-482 (2015).
  5. Z. Meng, A. J. Traverso, C. W. Ballmann, M. A. Troyanova-Wood, and V. V. Yakovlev, “Seeing cells in a new light: a renaissance of Brillouin spectroscopy,” Advances in Optics and Photonics 8, 300 (2016).
  6. G. Scarcelli, W. J. Polacheck, H. T. Nia, K. Patel, A. J. Grodzinsky, R. D. Kamm, and S. H. Yun, “Noncontact three-dimensional mapping of intracellular hydromechanical properties by Brillouin microscopy,” Nature Methods 12, 1132-1134 (2015).
  7. Z. Meng, S. C. Bustamante Lopez, K. E. Meissner, and V. V. Yakovlev, “Subcellular measurements of mechanical and chemical properties using dual Raman-Brillouin microspectroscopy,” Journal of Biophotonics 9, 201-207 (2016).

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