Performing Spectroscopy on Plasmonic Nanoparticles with Transmission-Based Nomarski-Type Differential Interference Contrast Microscopy

Summary

The goal of this protocol is to detail a proven approach for the preparation of plasmonic nanoparticle samples and for performing single particle spectroscopy on them with differential interference contrast (DIC) microscopy.

Abstract

Differential interference contrast (DIC) microscopy is a powerful imaging tool that is most commonly employed for imaging microscale objects using visible-range light. The purpose of this protocol is to detail a proven method for preparing plasmonic nanoparticle samples and performing single particle spectroscopy on them with DIC microscopy. Several important steps must be followed carefully in order to perform repeatable spectroscopy experiments. First, landmarks can be etched into the sample substrate, which aids in locating the sample surface and in tracking the region of interest during experiments. Next, the substrate must be properly cleaned of debris and contaminants that can otherwise hinder or obscure examination of the sample. Once a sample is properly prepared, the optical path of the microscope must be aligned, using Kohler Illumination. With a standard Nomarski style DIC microscope, rotation of the sample may be necessary, particularly when the plasmonic nanoparticles exhibit orientation-dependent optical properties. Because DIC microscopy has two inherent orthogonal polarization fields, the wavelength-dependent DIC contrast pattern reveals the orientation of rod-shaped plasmonic nanoparticles. Finally, data acquisition and data analyses must be carefully performed. It is common to represent DIC-based spectroscopy data as a contrast value, but it is also possible to present it as intensity data. In this demonstration of DIC for single particle spectroscopy, the focus is on spherical and rod-shaped gold nanoparticles.

Introduction

Since the 1980s, differential interference contrast (DIC) microscopy has largely been viewed as an important imaging method reserved for microscale objects within the biological sciences. However, during its development in the 1950s and 1960s, it was intended as a technique for materials science¹. With the recent advancements in the material sciences related to plasmonic nanoparticles, an increased interest in the characterization of materials with optical microscopy has taken place.

Many optical techniques are certainly available for nanomaterial characterization (e.g., dark field, brightfield, polarized light, fluo....

Protocol

1. Sample preparation with standard glass microscopy slides

1. Prepare glass microscope slides for sample deposition.
   NOTE: In some circumstances, it may be more appropriate to store the glass in ultrapure water instead of ethanol. However, storing in water or air makes the glass hydrophobic over time.
   1. For best results, purchase glass or quartz microscope slides and cover glass.
   2. Using a scribing pen, place a shallow and short scratch mark onto the center of each glass cover slip.

Discussion

When imaging with DIC microscopy, it is critical to optimize the optical components before collecting data. Even minor adjustments to the polarizer in the middle of an experiment can result in significant impacts to the final data⁶. Moreover, different materials require different polarizer settings. Although large step sizes were utilized here to demonstrate the effect of polarization angle, in an actual experiment, it is imperative to optimize the polarizer setting within 1°–2° of.......

Materials

Name	Company	Catalog Number	Comments
Contrad 70	Decon Labs, Inc.	1002	For cleaning microscope glass, Available through many chemical suppliers
Ethanol	Fisher Scientific	A962-4	For cleaning and storing microscope glass
Glass microscope cover slips	Ted Pella	260148
Glass microscope slides	Ted Pella	26007
Gold nanorods	Nanopartz	DIAM-SPR-25-650
Gold nanospheres (80 nm)	Sigma Aldrich	742023-25ML
ImageJ	NIH	N/A	Free Software availabe for data analysis from NIJ
Nail polish	Electron Microscopy Sciences	72180
Nikon Ti-E microscope	Nikon	N/A
Nitrogen gas	Airgas	N/A
ORCA Flash 4.0 V2+ digital sCMOS camera	Hamamatsu	77054098
Scribing pen	Amazon	N/A	Many options available online for under $10. Not necessary to buy an expensive version.
Ultrapure water			18 megaohm