Tissue refractometry using Hilbert phase microscopy

Abstract

We present, for the first time to our knowledge, quantitative phase images associated with unstained 5 μm thick tissue slices of mouse brain, spleen, and liver. The refractive properties of the tissue are retrieved in terms of the average refractive index and its spatial variation. We find that the average refractive index varies significantly with tissue type, such that the brain is characterized by the lowest value and the liver by the highest. The spatial power spectra of the phase images reveal power law behavior with different exponents for each tissue type. This approach opens a new possibility for stain-free characterization of tissues, where the diagnostic power is provided by the intrinsic refractive properties of the biological structure. We present results obtained for liver tissue affected by a lysosomal storage disease and show that our technique can quantify structural changes during this disease development.The light scattering by tissues is fully determined by the three-dimensional refractive index distribution associated with the biological structure [1-4] On the other hand, the refractive properties of tissues reflect their structural organization, which can be used as an intrinsic marker for disease. However, due to its inhomogeneous distribution in all three dimensions, the tissue refractive index is extremely difficult to measure directly. Highly scattering tissue has been characterized in terms of an average refractive index by optical coherence tomography [5] and, more recently, total internal reflection [6]. Recently, we applied quantitative phase imaging to live cells flowing in microfluidic devices, which provides a high-throughput method for cytorefractometry [7].In this Letter, we present a direct method for measuring the refractive index of biological tissues. The method extends, for the first time to our knowledge, the concept of quantitative phase imaging [8][9][10][11][12] to unstained tissue sections that are relevant for pathology and can be characterized as transparent, i.e., weakly absorbing and scattering objects. To measure these optical path-length maps, we employ Hilbert phase microscopy (HPM), which was developed in our laboratory for measuring quantitative phase images of cells with high lateral resolution and low noise [9,10].

Single-shot slightly-off-axis interferometry based Hilbert phase microscopy of red blood cells

“…In addition, the second phase peak in Fig. 6 (d) has a symmetrical shape which matches the previous literature [25]. …”

Biomedical applications of holographic microspectroscopy [Invited]

“…Topography and its dynamics have been widely addressed, including imaging of human tissues and studying pathophysiology of live biological cells [41][42][43][44]. Holographic imaging techniques measure the optical light field Ex; y; λ Ax; y; λ· exp−iΔϕx; y; λ, consisting of amplitude Ax; y; λ and phase delay maps Δϕx; y; λ, which is diffracted by samples at a specific wavelength of light λ.…”

Tissue refractive index as marker of disease

“…[30][31][32] It has been shown that the knowledge of the amplitude and phase associated with an optical field transmitted through tissues captures the entire information regarding light-tissue interaction, including scattering properties. [33][34][35][36] Yet, the potential of QPI for label-free pathology has not been explored. Here we employ spatial light interference microscopy (SLIM), [37][38][39] a new white light QPI method developed in our laboratory, to image the entire unstained prostate and breast biopsies and perform a side-by-side comparison with stained pathological slides.…”