Principles of microspectroscopy (2) Types and characteristics of microspectroscopy

November 24, 2020

Many traditional spectroscopies have been adapted to microspectroscopy. Techniques widely used in microspectroscopy of samples are ultraviolet-visible absorption, fluorescence, Raman scattering and infrared absorption (Table 1). The ultraviolet-visible absorption method measures absorbance of a sample and is suitable for obtaining a color spectrum in the visible region. In the fluorescent probe method, light emission by fluorescent molecules under excitation light is measured. This method may require fluorescence staining of the sample. Infrared absorption by a sample are measured using infrared absorption based on molecular vibrations, which are unique to every compound. Raman scattering measures molecular vibrations similar to infrared but using lasers in the visible and near-infrared regions. A Raman scattering microscope has high spatial resolution but the signal intensity is low, so that measurements can require a long time. Although an infrared absorption microscope has high sensitivity, it is difficult to obtain high spatial resolution. Raman and infrared are considered complimentary techniques.

Table 1. Types and features of microspectroscopy

Type Principles Phenomena Uses Features
Ultraviolet-visible Electronic transitions Transmission / reflection Optical materials Hue of observed image matches visible spectrum
Color analysis
Fluorescence Electronic transitions Emission Autofluorescence Visualizing localization of luminescent molecules with high contrast
Fluorescent probes
Quantum yield
Infrared Molecular vibrations Transmission / reflection Foreign matter Analysis of effects of factors such as temperature, electric field and light irradiation on sample state
Multilayer films / film thickness
Device failure
Reaction process
In-plane distribution
Component distribution
Raman Molecular vibrations Raman scattering Fine particles Using a confocal optical system, 3D imaging is possible by performing a scanning analysis in the depth direction.
Buried foreign matter
Silicon stress
Crystal polymorphs
Compound semiconductors
Inorganic materials
Pharmaceuticals / cosmetics

Spatial resolution in microspectroscopy

There are two types of objective optics for microspectroscopy: visible objective lenses and Cassegrain mirrors, and the type used depends on the purpose (Figure 1). For UV-Vis, fluorescence, and Raman objectives found in common microscopes may be used.  For FTIR Cassegrain objectives are required. Spatial resolution is important to microspectroscopy since it determines how small of features you can see.  In the case of an objective lens, the spatial resolution is 0.61 × λ/N.A., and for a Cassegrain mirror, it is 2.2 × λ/N.A., where N.A. is the numerical aperture and λ is the wavelength of light. Thus, the spatial resolution is theoretically higher for an objective lens. Spatial resolution also increases with decreasing wavelength. Table 2 summarizes the minimum observable area for different microspectroscopy methods.

Figure 1. Objective lens and Cassegrain mirror

Table 2. Types of microspectroscopy and observable areas

Type Condensing system Minimum size Issues Features
Ultraviolet-visible/fluorescence Cassegrain mirror / objective lens 10 μm Absorption / staining
Infrared Cassegrain mirror 5 μm Sample thickness Thin film preparation
Use of ATR
Raman Objective lens 1 μm Fluorescence Excitation wavelength selection
Fluorescence removal

Mapping and imaging

Creating a spectroscopic image of a sample may be done by either mapping or imaging a sample. First, a visual image of the sample must be obtained. In recent microspectroscopy systems, visual CCD imaging is performed instead of visual observation with an eyepiece. The magnification of the image is determined by the objective lens, and the sample position is adjusted by an XY stage to set the field of view. Mapping is performed by automatically scanning a target area in the visual image and performing measurements at multiple points. These points form individual pixels in a spectroscopic image. Imaging can also be performed by simultaneously measuring multiple points using a linear array CCD detector for UV-Vis, fluorescence, and Raman and a linear array MCT for FTIR (Figure 2). The spectroscopic image can be formed based on selecting a desired wavelength and examining the intensity of the peak at the points on the image.

Figure 2. Mapping vs. Imaging