What is FTIR Microscopy?

FTIR Microscopy

FTIR is concerned with the vibration of molecules.  Each functional group has its own discrete vibrational energy which can be used to identify a molecule through the combination of all of the functional groups. This makes FTIR microscopy ideal for sample ID, multilayer film characterization, and particle analysis.

FTIR Spectroscopy

FTIR is concerned with the vibration of molecules.  Each functional group has its own discrete vibrational energy which can be used to identify a molecule through the combination of all of the functional groups. This makes IR Microscopy ideal for sample ID, multilayer film characterization, and particle analysis. More can be found here on the fundamentals of FTIR Spectroscopy.

Reflection Measurements of FTIR Microscopy

Reflection measurement is performed with a Cassegrain objective, which is different from a regular microscope objective.  The Cassegrain objective (Figure 1) uses a primary and secondary mirror to send the IR beam onto the sample at 35o. IR reflective materials work best with this objective, while dark samples do not work as well.

Optical configuration of a Cassegrain objective for reflectance.
Figure 1. Optical configuration of a Cassegrain objective for reflectance.

Transmission Measurements of FTIR Microscopy

Transmission analysis of samples is probably the most common and most universal method of analyzing solids, liquids, and gases. Transmission measurements provide the highest sensitivity and best detection of all infrared sampling techniques. For transmission, two Cassegrain objectives are employed: focusing and condenser (Figure 2).  The focusing works the same as the one for reflection.  Light passes through the sample at the focal point and then hits the condenser which collimates the beam into the detector. Samples must be thin (less than 50 microns) and need to be in either a KBr pellet or diamond anvil cell. The focal planes of the Cassegrains are matched. Fibers, laminates, and thin films are often measured in transmittance mode.

Optical configuration of two Cassegrain objectives for transmission measurements.
Figure 2. Optical configuration of two Cassegrain objectives for transmission measurements.

ATR Measurements with FTIR Microscopy

Attenuated total reflectance (ATR) uses special crystal materials in contact with a sample to get chemical information.  The ATR (Figure 3) consists of focusing mirrors to hit the crystal at a 45o angle.  The light then passes into the sample and reflected back into the spectrometer.  To protect the objective, a pressure plate is required.  Over pressure can damage some crystals, with the exception of diamond objectives.  Diamond, ZnSe, and ZnS can be configured such that the sample can be seen when in contact with the sample.

Optical configuration of an ATR objective
Figure 3. Optical configuration of an ATR objective.

Grazing Angle

Coatings on ‘shiny’ substrates are excellent candidates for infrared reflection-absorption studies. As the coating gets thinner, incidence and collection angles can be varied from 45-75 degrees until the ‘grazing’ angle is reached, generally considered to be 85 degrees (Figure 4). The term ‘reflection absorption describes the progress of the incident beam as it passes through the coating, reflects from the substrate and passes through the coating again before reaching the detector. As the incident and collection angles approach grazing angle, the incident beam strikes the coating at shallower angles and the pathlength through the sample gets longer, enhancing the absorption intensity. Grazing angle reflection is used to examine the thinnest of surface coatings by ‘grazing’ the sample at a very shallow angle, the resulting longer sample pathlength providing greater sensitivity. Infrared reflection-absorption spectroscopy (IRRAS) is often used to study monolayer coatings on metals and other substrates. Limited quantitation can be made when evaluating the composition and thickness of the coatings.

Optical configuration of a grazing angle objective.
Figure 4. Optical configuration of a grazing angle objective.

Detectors

The many detectors that can be used in an FTIR microscope cover a wide wavelength range from the visible – (silicon photodiodes), NIR – (InSb or InGaAs), mid-IR – (TGS or MCT), far-IR – (Si bolometer). The IRT Series has a choice of standard detectors with the option of a second detector. The simplest detector, a Peltier cooled DLaTGS detector is used in the mid-IR region with good sensitivity, however, with much greater sensitivity an LN2 cooled mid-band MCT detector is better for measuring smaller microscopic areas. The optional second detector can be installed with a choice of either a fixed detector or a cassette system for interchangeable detectors, both can be selected from a wide range of options. To increase speed, especially for imaging or dynamic measurement a 16 element linear array detector (MCT or InSb) dramatically improves throughput (Figure 5.).

Illustration of mapping using a point detector (left) and a linear array (right).
Figure 5. Illustration of mapping using a point detector (left) and a linear array (right).

IQ Mapping

IQ Mapping is a unique feature that allows the measurement area to be moved in a stationery Cassegrain to build up an image map without moving the stage. IQ Mapping can be used with transmission, reflection and ATR measurements. It is exceptionally useful for ATR measurement as an area in contact with the crystal can be mapped without lifting and repositioning the objective prism, which normally causes damage to the surface during the process. IQ Mapping also provides imaging for soft samples, like gels or even viscous liquids. The ATR prism will agitate the sample if it is lifted and repositioned, but using the Clear View SS type ATR objective allows the sample to be viewed and measured without disturbing the sample once it is in position.

Fluorescence Observation in FTIR Microscopy

Fluorescence observation can be used to identify fluorescent samples which cannot be seen with visible light (Figure 6.). Two options are available with different wavelength ranges. The wavelength range is selected with a filter.

Fluorescence of polymer beads at different wavelengths.
Figure 6. Fluorescence of polymer beads at different wavelengths.

Differential Interference Contrast Observation (DIC)

Differential Interference Contrast Observation (DIC) uses polarized light and a Nomarski-modified Wollaston prism to enhance the observation of images with low contrast. DIC uses phase difference in light to stereographically view very small step differences in the submicron order. Nomarski prisms are used to create bright and dark contrast from the differences in the two beams directly reflected at the sample’s surface. This technique can be applied equally to low contrast biological and non-biological samples that have small unevenness in the surface, an example may be seen in Figure 7.

Bright field and DIC image of a biological material.
Figure 7. Bright field and DIC image of a biological material.

Visible Polarization

Polarized light observation (PLO) exploits the differences in anisotropic properties to enhance the observation of materials with low contrast. PLO uses two polarized elements located in the optical path on each side of the sample being observed. It is particularly useful for samples such as biomolecules and biostructures, minerals, ceramics, mineral fibers, extended polymers, liquid crystals, etc.  DIC of a polymer sample may be seen in Figure 8.

Unpolarized (left) and Polarized view (right) of a polymer sample.
Figure 8. Unpolarized (left) and Polarized view (right) of a polymer sample.

FTIR Microscopy Webinar

This introductory webinar covers FTIR imaging. After a quick recap of FTIR theory and instrumentation, sample preparation, techniques and objectives are discussed. Additionally, hardware such as sample stages, detectors, and complimentary microscopy techniques are reviewed.

Powerpoint slides can be downloaded here.

Applications of FTIR Microscopy

Microplastics Identification by IR Imaging Measurement

The objective of this application note is to demonstrate IR microscopy as an effective and rapid assessment tool to identify and characterize microplastics in water. Using IR microscopy, microplastics can be measured without elaborated pretreatment, and the type of plastic can be easily identified using FTIR databases. In addition, using IR imaging, it is possible to calculate the distribution ratio and measure the particle size.

FTIR Microscopy for the Analysis of Polymer Laminates in Beverage Packaging

In this application note, infrared spectroscopy provides the ability to study the interactions of the vibrational and rotational energies of atoms or groups of atoms within molecules. Infrared spectra reflect vibrational motions that produce a change in the permanent dipole moment of the molecule. Infrared spectroscopy is a powerful qualitative and quantitative tool. There is a large amount of information that can be gained by using infrared spectroscopy for the analysis of polymers. Minor changes to the molecular structure usually result in a spectrum that is clearly distinguishable from the spectrum of the original compound. Thus, they can be used to identify the presence of a specific chemical compound or mixture of compounds.

Secondary-Structure Analysis of Proteins using FTIR Imaging 

In this application note, we performed infrared imaging on slices of a mouse eyeball using the IMV-4000. We analyzed the IR spectra of each using primary component regression (PCR) and created an in-plane distribution map of the secondary structure of proteins (Figure 1). The measured area was 2750 x 1600 µm and the measurement size was 12.5 x 12.5 µm since a 16X cassegrain objective was used. 128 x 220 (28,160) points were measured. There were four integrations and the spectral resolution was 8 cm-1. The slice was placed on BaF2 (10mm diameter and 1mm thick) and measured in transmission. Figure 1-(1) shows an H&E (Hematoxylin and eosin)-stained image of the slice and Figure 1-(2) shows a visible image of the measurement area.