Measurements of complex refractive index change of photoactive yellow protein over a wide wavelength range using hyperspectral quantitative phase imaging

Kyeoreh Lee,Hyotcherl Ihee,Youngmin Kim,Jaehwang Jung,Yongkeun Park

doi:10.1038/s41598-018-21403-z

Abstract

A novel optical holographic technique is presented to simultaneously measure both the real and imaginary components of the complex refractive index (CRI) of a protein solution over a wide visible wavelength range. Quantitative phase imaging was employed to precisely measure the optical field transmitted from a protein solution, from which the CRIs of the protein solution were retrieved using the Fourier light scattering technique. Using this method, we characterized the CRIs of the two dominant structural states of a photoactive yellow protein solution over a broad wavelength range (461–582 nm). The significant CRI deviation between the two structural states was quantified and analysed. The results of both states show the similar overall shape of the expected rRI obtained from the Kramers–Kronig relations.

Highlights

Light-matter interaction is key to the characterization of a sample and is performed using a diverse range of existing methodologies, including visual inspection, scattering analysis, microscopy, and spectroscopy
Simultaneous complex refractive index (CRI) measurement would be highly preferable for protein studies with the capability of performing spectroscopic measurements because the proteins are sensitive to surrounding environment such as temperature, pH, and ion concentrations, which may be perturbed during each individual measurement[10,11,12,13]
In order to obtain the CRI of photoactive yellow protein (PYP), we first measured the multi-wavelength light field images of a 100-μm-diameter polymethyl methacrylate (PMMA) microsphere immersed in PYP solution (Fig. 1)

Summary

Introduction

Light-matter interaction is key to the characterization of a sample and is performed using a diverse range of existing methodologies, including visual inspection, scattering analysis, microscopy, and spectroscopy. Circular dichroism spectroscopy, which analyses the CRI difference between the two circular polarizations of light, has been utilized to determine the secondary structures of proteins[8] and ultraviolet-visible spectrophotometry, which measures the imaginary part of the CRI (molar extinction coefficient) has been utilized to determine the protein concentration or property[9]. Despite their powerful molecular characterization capabilities, direct measurement of the CRIs of protein solutions over a wide range of wavelengths has been hindered by limitations in existing instruments. Because the light scattering information is highly sensitive to the CRI of the surrounding medium, the CRI of the protein solution can be calculated precisely from the light scattering information using Mie theory[34]

Methods

Results

Discussion

Conclusion