Targeted Alteration of Real and Imaginary Refractive Index of Biological Cells by Histological Staining

Abstract

Various staining techniques are commonly used in biomedical research to investigate cellular morphology. By inducing absorption of light, staining dyes change the intracellular refractive index due to the Kramers-Kronig relationship. We present a method for creating 2-D maps of real and imaginary refractive indices of stained biological cells using their thickness and absorptance. We validate our technique on dyed polystyrene microspheres and quantify the alteration in refractive index of stained biological cells. We reveal that specific staining of individual organelles can increase their scattering cross-section by orders of magnitudes implying a major impact in the field of biophotonics.Staining provides a tool for visualization of macromolecules, biological cells, and tissues in applications that extend from basic biology to clinical diagnostics [1,2]. Within the field of optics, the change in light scattering properties of stained organelles caused by the absorption of specific dyes is used to determine their refractive indices [3]. However, while most of the attention has been focused on the absorption (i.e. the imaginary part of refractive index) introduced by staining dyes, the subsequent alteration in the real part of refractive index according to the Kramers-Kronig relation has often been overlooked [4,5]. A recently proposed linear relationship between the real and imaginary parts of refractive index does not treat the effects of spectral dispersion in either quantity [6]. In this Letter, we demonstrate a method to quantify the wavelength dependent change in both real and imaginary parts of the refractive index of epithelial cells caused by histological stains such as hematoxylin and eosin-containing cytostain.We construct 2-D maps for real and imaginary refractive indices (n' and n") of stained cells as a function of wavelength λ as follows: first, we calculate the n" after measuring the transmission intensity I and the sample thickness L using: (1) where I 0 (x,y,λ 0 ) is the transmission intensity at a wavelength λ 0 where absorption is absent (to isolate n" from other causes of attenuation). For dyes used in this study λ 0 = 700nm. Then, we extract the n' (λ):* Corresponding author: v-backman@northwestern.edu. where χ' dye +jχ" dye is the electric susceptibility of dye molecules embedded in a nonabsorbing material with a refractive index n 0 ; χ' dye is determined from χ" dye using Kramers-Kronig relations [4,5,7]: (3) where P denotes Cauchy principal value and k is the wavenumber k=2π/λ. Our instrumentation includes an atomic force microscope (AFM, Bruker Bioscope II) with 39 nm resolution to measure the thickness L(x,y) and a custom built spectroscopic microscope (SM) to image the sample, as well as measure the wavelength dependent transmission through it I(x,y,λ). The sample of interest is mounted between 2 glass slides using Permaslip (Alban Scientific) to remove specular reflections at the specimen-air interface. The bottom glass slide is coated with a 100nm layer of Aluminum (Deposit...

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