Eunah Lee

Optimizing Depth Resolution in Confocal Raman Microscopy: A Comparison of Metallurgical, Dry Corrected, and Oil Immersion Objectives

Spherical aberration is probably the most important factor limiting the practical performance of a confocal Raman microscope. This paper suggests some simple samples that can be readily fabricated in any laboratory to test the performance of a confocal Raman microscope under realistic operating conditions (i.e., a deeply buried interface, rather than the often-selected alternative of a bare silicon wafer or a thin film in air). The samples chosen were silicon wafers buried beneath transparent polymeric or glass overlayers, and a polymer laminate buried beneath a cover glass. These samples were used to compare the performance of three types of objectives (metallurgical, oil immersion, and dry corrected) in terms of depth resolution and signal throughput. The oil immersion objective gave the best depth resolution and intensity, followed by a dry corrected (60×, 0.9 numerical aperture) objective. The 100× metallurgical objective was the worst choice, with degradations of ∼5× and 8× in the depth resolution and signal from a silicon wafer, comparing a bare wafer with one buried under a 150 μm cover glass. In particular, the high signal level obtained makes the immersion objective an attractive choice. Results from the buried laminate were even more impressive; a 30× improvement in spectral contrast was obtained using the oil immersion objective to analyze a thin (19 μm) coating on a PET substrate, buried beneath a 150 μm cover glass, compared with the metallurgical objective.

Experimental Evaluation of the Depth Resolution of a Raman Microscope

Raman microscopy has been attractive because of its ability to characterize materials on a spatial scale commensurate with optical microscopy. Typically the lateral spatial resolution is quoted as determined by the Airy disc[1] which is 1.22λ/NA where λ is the wavelength of the illuminating light, and NA is the numerical aperture which is equal to nsinθ, where n is the index of refraction of the medium (1.0 in the case of air) and is the angle subtended by the optics. However, the Airy disc description cannot be correct for a Raman microscope. The Airy disc assumes uniform illumination of the focusing optic, and the laser profile is anything but. In addition, in some instruments the Gaussian laser profile is not well matched to the aperture of the focusing objective. At any rate, this article is going to concentrate on the depth resolution of the Raman microscope. Optical calculations for depth resolution of an optical microscope state that the it is proportional to λ/(NA). The essential point to recognize is that the spatial resolution of any Raman microscope depends on the detection optics as well as the focusing optics. How effectively does the optical system collect the Raman signal excited in the laser focal spot, and reject the signal from the surrounding volume that is illuminated by the laser but not in focus?

Improving and Understanding Three Dimensional Spatial Resolution in a Confocal Raman Microscopy and Raman Hyperspectral Imaging I

Confocal Raman microscopy provides a high spatial resolution because it operates in short wavelength region and utilizes confocal optics. However, the spatial resolution of a confocal Raman microscopy is not well understood, and often confused with the smallest measurable sample size. When performing Raman hyperspectral imaging with a confocal Raman microscope, it is also confused with the smallest distance a mapping stage can step. While all these parameters are pertinent to record a good Raman spectrum or a good Raman map, they are not spatial resolution, and thus have different impacts to the data and results. In this and subsequent papers, we will begin with the theoretical definition, examine the instrumental implementations and present the empirical applications of these parameters with examples.

Single Walled Carbon Nanotube Analysis by Photoluminescence and Raman Microscopy: Rapid, Robust Acquisition and Simulation of Quantum and Chiral Maps to Ease Structural Assignments

This paper describes improved methods for both data acquisition and analysis that are pertinent to interpreting the structural composition of single-walled carbon nanotube (SWNT) mixtures and suspensions. Rapid data acquisition of photoluminescence is made possible with a specially configured spectrofluorometer which uses a liquid nitrogen-cooled InGaAs array detector, imaging spectrograph, excitation reference photodiode and tunable xenon excitation source to collect seamless, instrument-corrected, excitation-emission quantum maps. The preferred instrument configuration with the InGaAs array can generate a quantum map with both high S/N levels and spectral resolution in only seconds to minutes; previous single-channel InGaAs photodiode and photomultiplier detector measurements took hours to days. The standard spectral range for excitation and emission is from 250 nm to 1700 nm with an option to extend to 2200 nm. Robust analysis of the quantum maps is facilitated by a custom global analysis program (US Patent Pending) which incorporates a powerful ‘double-convolution integral’ DCI algorithm to simultaneously model the excitation and emission spectral bands for each SWNT species. The patented DCI algorithm reduces the number of free fitting parameters for the spectral simulation by up to a factor of 1000 compared to conventional 2 D spectral simulators. The global analysis yields quantitative information on the chirality and diameter parameters of SWNT species in a given mixture. Together the acquisition hardware and analysis software constitute significant developments with respect to enhanced sensitivity and statistical significance for quantifying the components of complex SWNT mixtures. Raman maps of SWNTs were collected from dispersions plated onto substrates such as silicon plates using multiple excitation wavelengths. Identification of the chirality of the tubes was made using the Kataura plot. In many instances single lines in the radial breathing mode region were recorded indicating the presence of a single tube. The implication for this observation is that there is more than adequate sensitivity to detect and identify single-walled tubes. More recently Raman excitation on an AFM using a gold-coated tip to induce surface enhancement has been achieved. In this configuration it will be possible to map the topology of the tubes using detailed information on their structural composition.

A Comparison of Raman and EDXRF Chemical Imaging for Use in Formulation Process Development and Quality Control

Compounds of magnesium and calcium are common components of pharmaceutical formulations. Spectroscopic imaging, and in particular hyper-spectral imaging, can provide a complete understanding of a formulation. One of the excipients, magnesium stearate, which is often added because of its lubricating properties, can be difficult to observe visually. In this paper we will compare two spectral imaging techniques Raman Microscopy and Energy Dispersive X-Ray Fluorescence (EDXRF) Microscopy. These two techniques, Raman and EDXRF microscopy, provide molecular and elemental images respectively. Both analytical techniques are being used in process development and quality control. Data will be shown that highlights the capabilities of both techniques and discusses the advantages of each. Raman microscopy allows very high spatial resolution of molecular maps (1 μm); hyperspectral images very accurately show particle size and distribution in a formulation. The EDXRF microscope provides a rapid method to obtain hyperspectral elemental images with 10 μm spatial resolution. For both techniques little or no sample preparation is required.