Microscopic imaging ellipsometry of submicron-scale bacterial cells

Mai Ibrahim Khaleel,Ching-Hang Chien,Yia-Chung Chang,Yu-Da Chen

doi:10.4314/tjpr.v16i11.20

Abstract

Purpose: To demonstrate the power of microscopic imaging ellipsometry (MIE) to identify submicronscale bacterial cells and track their surface topology variation. Methods: Microscopic imaging ellipsometry with rotating compensator configuration was used to measure the ellipsometric spectra for dried submicron-scale Streptococcus mutans cells cultured on gold (Au) film for wavelengths (λ) in the visible range (λ = 490 – 710) nm. The ellipsometry characteristic images, Psi (Ψ), Delta (Δ), p- and s- polarized reflectance (Ip and Is), and reflectance difference image (RDI) for a chain of two and four dried cells, were collected for a series of different objective planes (near the focal plane) for λ = 600nm. Results : The results show that by adjusting the position of objective planes (POP) to achieve the best focus, it was possible to identify cells smaller than 1 μm and observe their diffraction patterns in Ψ and Δ images. It was observed that Δ spectra and images were particularly sensitive to POP, while Ψ spectra and images for dried S. mutans cells were rather insensitive to POP. Conclusion : MIE is a sensitive non-optical technique that can be used to image biological systems without the need for labeling of molecules. Keywords: Objective plane, Imaging ellipsometry, Submicron-scale bacterial cells, Streptococcus mutans, Biosensing, Focal plane

Highlights

Ellipsometry is an optical technique used to determine the thickness and optical constants of samples based on measuring two characteristic parameters, Ψ (Psi) and Δ (Delta), which describe the polarization contrast and phase change of light reflected from the sample [2]
We proposed microscopic imaging ellipsometry with rotating compensator configuration (MIE-RCE) as a sensing tool to detect the distribution of dried S. mutans cells on a glass substrate and determine their optical constants and thickness using theoretical models based on effective medium theory [14]
Ellipsometry spectra and images were measured for two and four dried S. mutans cells cultured on Au film at different objective planes in specular reflection mode for λ= (490 – 710 nm) at an AOI = 52o

Summary

Introduction

Ellipsometry is an optical technique used to determine the thickness and optical constants of samples based on measuring two characteristic parameters, Ψ (Psi) and Δ (Delta), which describe the polarization contrast and phase change of light reflected from the sample [2]. The technique is well known for being non-contact, non-destructive, and noninvasive [7,8,9] It does not require special sample preparation nor labeling of molecules, in comparison to other detection techniques that require labeling- usually enzymatic or fluorescent such as fluorescence imaging [9,10,11]. MIE provides submicron spatial resolution that cannot be achieved by IR/thermal imaging technique since it is based on detecting thermal radiation from the sample and not capable of extracting highresolution structure information as MIE does [14]. MIE can detect very thin layers and measure their precise thickness, while Raman spectroscopy is sensitive to the chemical nature of the sample [13]

Results

Discussion

Conclusion