A Multimodal Wide-Field Fourier-Transform Raman Microscope

Summary

A wide-field Fourier-transform microscope, based on a compact and ultra-stable birefringent interferometer, allows the parallel acquisition of spectra for all pixels of a 2D detector. The time-domain approach enables the disentanglement of photoluminescence and Raman signals, and allows rapid Raman mapping (~5 ms/pixel) with ~1-µm spatial and 23-cm^-1 spectral resolution.

Abstract

Raman microscopy is a powerful technique to determine the chemical composition of a sample. The main challenge of the state-of-the-art spontaneous Raman microscopes, based on point-scanning frequency-domain detection, is the acquisition of Raman maps of large areas due to typically long pixel dwell times (0.1-1 s). The wide-field Fourier-transform (FT) microscope presented here allows rapid measurement of extended samples with ~1-µm spatial and ~23-cm^-1 spectral resolution. It is based on an ultra-stable and compact common-path birefringent interferometer, added along the detection path of a commercial microscope, which enables the parallel acquisition of spectra for all pixels of a 2D detector, significantly reducing the measurement time (10-100x faster). The time-domain approach effectively addresses another limitation of the frequency-domain technique, which is disentangling the Raman signal from the photoluminescence background, in which the Raman signal may be buried. This protocol describes how to perform hyperspectral measurements with an upgraded commercial microscope: the alignment of the illumination scheme, the criteria to choose measurement parameters, and the data analysis to retrieve and investigate spectra.

Introduction

Spectral imaging is a technique that provides both spectral and spatial information and has been applied to various fields, from microscopy¹ to remote sensing². Multispectral and hyperspectral imaging are distinguished based on whether a few discrete spectral bands or a continuous spectrum are recorded for each pixel of a 2D sensor. Raman imaging is a particular implementation of spectral imaging.

Raman scattering microscopy is a powerful technique to measure vibrational frequencies ν_R of a system. Vibrational modes are associated with the chemical bonds, ....

Protocol

The reagents and the equipment used in this study are listed in the Table of Materials.

1. Sample preparation

CAUTION: Wear latex gloves during this preparation since CdS pigment is toxic, especially when it is inhaled.

1. Use a scraper to deposit 20 mg of each pigment powder on a precision balance, obtaining a 1:1:1 weight proportion.
   NOTE: Carefully clean the scraper tip for each sample to avoid cross-contamination.
2. Mix the powders with a mortar to remove clumps.
3. Pour the resulting mixture onto a microscope slide; use the scraper tip to gently ....

Results

Figure 4A shows the Raman map for a selected region of the powder mixture. It is a false-colour representation of the abundance maps retrieved with MCR-ALS, as described in step 4.5. It provides information about the sample composition, since the three species can be clearly distinguished within the FOV. Figure 4B shows the average spectra in the selected regions of the map, with a spectral resolution of 23 cm^-1 defined by the chosen scan length. The .......

Discussion

A wide-field FT multimodal microscope has been introduced for the acquisition of spontaneous Raman scattering and luminescence images, and it is applicable to materials science. The applicability to biological samples is demonstrated by Candeo et al.¹⁷, who presents a hyperspectral fluorescence image of stained human cells. The main challenge for Raman scattering measurements in this field is to prevent photodamage resulting from inefficient heat dissipation from a widely illuminated area. A .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anatase	Kronos	1200	Polymorph of White TiO2 pigment
Cadmium sulfide powder	Kremer Pigmente GmbH & Co.	21060	Cadmium yellow, colour index dark/PY35
Camera	Andor	Luca R	Electron multiplying CCD camera with 1004x1002 pixels of 8µm x 8µm
Clean-up filter	Semrock	LL01-532-12.5	Ø1/2" 2.0 nm- FWHM laser-line filter at 532 nm
Coverslips	BRAND GmbH & Co	4700 55	Borosilicate glass 22x22x0.15 mm microscope coverslips
Dichroic mirror	Semrock	Di02-R532-25x36	25.2x35.6 mm single-edge laser dichroic beamsplitter at 532 nm
Fiber	Thorlabs	M74L05	400µm-core size, NA 0.39 multimode fiber
Laser	Hubner Photonics	Cobolt Samba 1000	CW diode pumped laser at 532 nm, maximum power 1000 mW
Long pass filter	Semrock	LP03-532RU-25	Ultra steep long-pass edge filter at 532 nm
Microscope	Leica	DMRBE	Trinocular research microscope
Objective	Leica	PL-FLUOTAR, 10x/0.30	Excitation and collection objective with 10x magnification, 0.3 NA
Objective	Newport	M-10X	Objective lens in illumination path, with 10x magnification, 0.25 NA
Rutile	Kronos	2900	Polymorph of White TiO2 pigment
Short pass filter	Thorlabs	FESH600	Ø25.0 mm shortpass filter with cut-off wavelength 600
Slides	Marienfeld	1000612	76x26x1 mm microscope slides