Spectroscopy and microscopy are great tools for analysis individually, but when combined they can yield even more powerful results. Spectroscopy shows the interaction between electromagnetic radiation and matter. The interactions give rise to electronic excitations, molecular vibrations or nuclear spin orientations. Microscopy allows amplification of samples that are too small to be seen by the naked eye.
Combining the two allows for the inspection of structures particles close to the limits of optical resolution and allows for a 3D view of samples. The microscope and spectrometers are linked via an optical fibre that is coupled to the microscope by a special optical adapter.
There are many different types of spectroscopy, but those known to be able to be coupled with microscopy include Infra-red (IR) spectroscopy, Raman spectroscopy and X-Ray spectroscopy.
Infrared (IR) spectroscopy analyses compounds using the infrared spectrum. The IR spectrum is split into near IR, mid IR and far IR, with near IR having the greatest energy. Near IR can penetrate a sample much deeper than far or mid IR, but it is also the least sensitive.
IR spectroscopy works by passing a beam of IR light through a sample, and the molecules of the sample vibrate when they absorb infrared radiation. The bonds stretch and bending, and when the frequency of the IR is the same as the vibrational frequency of the bonds, absorption occurs and a spectrum is recorded.
Raman spectroscopy is a type of vibrational spectroscopy that is similar to IR, but it uses inelastic scattering. Raman detects inelastic scattering, also known as Raman scattering, of monochromatic light from a laser in the visible, ultraviolet or near infrared range.
Raman spectroscopy provides a molecular fingerprint of the chemical composition and structures of samples, but the scattering gives inherently weak signals and is enhanced with techniques such as Surface Enhanced Raman Spectroscopy (SERS).
X-ray Spectroscopy uses X-ray excitation to promote electrons to higher energy levels. The absorption or emission energies gained from excitation are emitted as electrons, and these electrons have wavelengths which are characteristic to the elements of interest. X-ray absorption and emission spectroscopy is used to give qualitative results that determine bonding and elemental composition of the sample.
The main types of microscopy that can be combined with spectroscopy are optical microscopy, electron microscopy and scanning probe microscopy.
Optical microscopy is also known as light microscopy, and it was the first type of microscopy discovered. The microscope contains refractive glass and multiple lenses which produce enlarged images of samples when they are in the focal plane.
Optical microscopes can be split into categories based on their design, such as compound microscopes, stereo microscopes and confocal microscopes. Optical microscopes can also have ultraviolet, IR and fluorescence filters attached for a more in-depth analysis of samples.
Fluorescence microscopy is a type of optical microscopy that was developed later as the need to analyse fluorescent probes increases. Fluorescently labelled proteins and chemical stains are commonly used in biological research to study proteins and cells. The sample is illuminated with light ranging from the ultraviolet to the visible wavelengths, which is absorbed by the fluorophores in the sample, causing them to emit light of longer wavelengths to make up an image.
Electron microscopy uses an electron beam to create an image, with electromagnets acting as lenses. It has a much higher resolution than optical microscopy due to the wavelength of the electrons being much smaller than that from a bulb or laser, allowing for greater detail when scanning.
Electron microscopy can be split into transmission electron microscopy (TEM) and scanning electron microscopy (SEM). TEM works by sending a beam of electrons through a very thin sample and captures the electrons that have passed through to create a highly detailed two-dimensional image. SEM works by sending a beam of focused electrons to the sample and bouncing them off, which creates a three-dimensional surface image. TEM is used to study the interior of a sample, whereas SEM is used to study the surface of a sample.
Scanning probe microscopy scans the surface of samples with a probe to measure fine surface shapes and properties and generate an image. The types of scanning probe microscopy include atomic force microscopy (AFM), scanning tunnelling microscopy (STM) and near-field scanning optical microscopes (MSOM).
An AFM has a fine silicon or silicon nitride probe attached to a cantilever. STMs have a metal tip with a single apical atom, with the tip being attached to a tube where the current flows. MSOM has a probe that consists of a light source in an optical fibre, which is covered with a tip. The disadvantages of using scanning probe microscopy techniques are that they are slow, and the maximum image size is limited.
The most common types of spectroscopy to be combined with microscopy are Raman spectroscopy and IR spectroscopy, but developments in ultraviolet/viable and X-ray spectroscopy have also allowed them to also be linked microscopes.
IR microscopy can be split into Fourier Transform IR (FTIR) microscopy and scanning tunnelling microscopy. FTIR microscopy uses light microscopy to locate the region of interest in a sample, and then then it is non-invasively and non-destructively sampled with IR to provide chemical identification.
IR spectroscopy can be coupled with STM microscopy to use the spatial resolution of STM with the vibrational information of IR spectroscopy. STM is used as a sensitive detector that can probe the IR response of sub-monolayers of molecules on conducting crystals for samples that are adhered to a surface.
Raman spectroscopy can be combined with confocal microscopy, atomic force microscopy and scanning electron microscopy. Confocal Raman spectroscopy is a non-invasive optical method to obtain detailed information about the molecular composition of samples. Confocal Raman spectroscopy yields chemical information about nano-materials, as it can use to sub-micron spatial resolution. Integrating Raman spectroscopy with AFM gives both the chemical characterization of Raman, and the topographical, thermal, mechanical, electrical, and magnetic results of AFM with molecular resolution.
A type of SEM Raman known as RISE (Raman Imaging Scanning Electron) uses confocal Raman and scanning electron (SEM) techniques in an integrated system. The blending of the two techniques allows for nanometer-scale visualization of surfaces from SEM combined with molecular detection specificity of Raman spectroscopy. The confocal Raman microscope is integrated into the vacuum chamber of the electron microscope and non-destructive SEM and Raman measurements are performed consecutively inside the chamber.
Two final spectroscopy and microscopy pairings that are not as widely used include linking X-Ray Spectroscopy and Shear Force Microscopy, and the ultraviolet (UV)/vis micro spectrometer. X-Ray Spectroscopy can be combined with microscopy as another way of simultaneously obtaining the surface topography of a sample as well as and chemically mapping it. The UV/vis micro spectrometer combines an optical microscope with an optical spectrophotometer to measure transmittance, absorbance, fluorescence, polarization, photoluminescence and reflectance of a sample.