Table of ContentsIntroduction
FT-IR as an Imaging System
ResolutionIR Analysis of a Wheat StemApplicationsAbout Bruker Optics
microscopy is a well-established method for the chemical identification of
particles or contaminants and for visualizing the distribution of certain
substances in complex compounds. Due to the usage of modern focal plane array
detectors, this technology has advanced to a new imaging technique during the
last few years. It allows for the measurement of even large sample areas with a
very high lateral resolution within a few minutes.
FT-IR as an Imaging System
After the measurement, the results are evaluated on the basis of easily
comprehensible images. For more than twenty year, even very small samples have
been analyzed using Fourier transform infrared (FT-IR) spectroscopy. For
this purpose, microscopes having mirror optics have been developed that allow
not just a visual viewing but also for infrared spectroscopic analysis of a
sample. Recently, FT-IR microscopes equipped with focal plane array (FPA)
detectors consisting of a matrix of 16 x 16 up to 128 x 128 detector elements
have been deployed. This new array detector allows the user to acquire up to
16,000 pixels/spectra simultaneously and has therefore enabled the FT-IR
microscopy to become also an imaging technique.
microscopy just as in other types of optical microscopy, the lateral
resolution is limited by the light diffraction. The smallest distance (δ) at which two points of a sample can still be separated is
described by the following formula:
δ = 0,61 λ / NA,
with NA being the numerical aperture of the objective and λ being the wavelength of the light.
Since the numerical aperture of the mirror objectives in FT-IR microscopes is
about 0.6, the above formula can be reduced as follows:
Distance δ = Wavelength λ.
Figure 1 shows the visible image of a sample which consists of a regular
pattern of metal strips on glass.
Figure 1. Pattern of metal strips on glass. The upper a)
image is a video image of the sample. The video image covers a sample area of
about 400 x 500μm. This sample area has been measured
in the reflectance mode with a pixel resolution of 1,1 μm using the FT-IR imaging system BRUKER HYPERION 3000
equipped with a FPA detector (64 x 64 detector elements) and a 36x objective
(NA=0.5). In b the IR image at 1650cm -1 (@
6 μm wavelength) is shown, in c the IR image at
3200cm-1 (@ 3 μm
The strips are arranged in groups of parallel oriented strips of different
sizes and separated by different wide gaps. The sample area of about 400 x
500μm as shown in figure 1 has been measured in
transmittance mode with a pixel resolution, which is the size of the sampling
area which is imaged on one detector pixel of 1.1 mm
using the FT-IR
imaging technique. The intensity of the spectra at the wavelengths of
1650cm-1 (@ 6 μm;
fig 1b) and 3200cm-1 (@ 3 μm; fig 1c) are displayed in false color.The two false color
images show that the rectangular form of the metal strips is made sharper and
that the groups of the smaller strips are resolved better at a wavelength of 3
μm than at a wavelength of 6 μm. This result shows that the lateral resolution of a well
imaging system is only restricted by the light diffraction.
This fact becomes
more obvious when you have a closer look at the cut along the red lines in group
6 and 7 marked in Figure 1c. The strips in group 6 separated by 14 to 8 μm are well-resolved at either wavelengths, 3 μm and 6 μm as shown in Figure 2.
However if the strip pattern and the gaps between the individual strips become
smaller, as in group 7, the lateral resolution achieved at a wavelength of 3 μm is much better than at a wavelength of 6 μm. According to the theoretical expectations, it was found
that structures can be resolved up to 6 μm at a
wavelength of 6 μm. And in case of a wavelength of
3μm, the resolution limit was shown to be about 3μm.
This high spatial resolution has been achieved because the used pixel
resolution, which is equal to the edge length of each detector element of 1,1
μm is considerably higher than the structures to be
resolved, i.e. all strips and gaps have been imaged on several pixels and not on
only one single pixel as shown in Figure 2.
Figure 2. Resolution profile. This figure shows a cut
through group 6 and 7 according to the red lines drawn in figure 1c. The blue
trace corresponds to the profile at 3200 cm-1 and the red trace
corresponds to the profile at 1650 cm-1.
IR Analysis of a Wheat Stem
The study of animal and vegetable tissues is also a standard application for
imaging. Figure 3 and 4 show the resulting data for a wheat stem tissue. The
main biochemical components such as proteins, carbohydrates and waxes/lipids can be
detected by their characteristic absorption. The IR images in Figure 4 show the
distribution of these components within the tissue. This information can also be
combined into a RGB image as shown in Figure 3 proving the distribution of
proteins (green), carbohydrates (red) and waxes/lipids (blue) in parallel.
Figure 3. Wheat Stem. In a the video image of a wheat
stem (microtome section; thickness of 10 μm) is shown .
The video image covers a sample area of about 500 x 500 μm. The RGB image (b) shows the combined distribution of the
proteins (green), carbohydrates (red) and the waxes/lipids (blue).
Figure 4. Wheat stem sample (see figure 3) area has been
measured in transmittance with a pixel resolution of 2.7 μm using the FT-IR imaging system BRUKER HYPERION 3000
equipped with a FPA detector (64 x 64 detector elements) and a 15x objective
(NA=0.4). The individual IR images show the distribution of proteins (1710 -
1480cm-1, a), carbohydrates (1140 – 900 cm-1, b) and
waxes/lipids (1770 – 1700 cm-1, c) within the tissue. The
distribution of these components has been visualized by evaluating the intensity
of the characteristic vibrations of each of these three biochemical components.
microscopy can be used for a broad range of applications. Typical
applications are the chemical identification of particles and smallest
contaminations, the examination of the homogeneity of coatings and the analysis
of the distribution of a multitude of different components in a complex mixture.
An important benefit of this technique is that it can be used to analyze nearly all kinds of samples. Moreover, it is a non-invasive method that does not require
staining or labeling of the sample under examination. The molecules are
identified on the basis of their characteristic vibrations which they are
excited by the impinging IR beam. In this way, FT-IR imaging
delivers information about the molecular composition of the analyzed sample
area. Hence this image technique is also called ‘chemical imaging’.
About Bruker Optics
Optics, part of the Bruker Corporation is the leading manufacturer and
worldwide supplier of Fourier Transform Infrared, Near Infrared and Raman
spectrometers for various industries and applications. their product line
includes FT-IR, NIR, Raman, TD-NMR, TeraHertz spectrometers and imaging
spectrographs for various markets and applications.
This information has been sourced, reviewed and adapted from
materials provided by Bruker Optics.
For more information on this source, please visit Bruker