FTIR Microspectroscopy
Fourier Transform Infrared Spectroscopy is a workhorse of the industrial materials
testing division. The combination of a microscope with an infrared spectrometer results
in a powerful instrument that allows for the detection of chemical species from a specific
spatial region.
Combining spatial specificity with information on its chemical constitution, a chemical
species map may be constructed for the whole spatial area. This typically involves
collecting the IR spectrum of the sample at a point, moving the sample to another
location and collecting the spectrum at the second point ans so on. In this manner, the
whole area is mapped point-by-point.
The Concept of FTIR Imaging
The state of the art in FTIR microspectroscopic instrumentation today is the combination
of a Focal Plane Array (FPA) detector with a step-scan spectrometer. This configuration
allows for the imaging of a wide field of view in a single collection. The concept of
imaging using an FPA is illustrated with a typical example in the figure below. The beam
of a step-scan spectrometer is diverted through standard microscope optics with the FPA
at the end of the optical train.
The premise behind the imaging experiment is that each pixel on the FPA corresponds to
a unique spatial region on the sample. The time required to collect a spectrum for a single
pixel is the time required to collect the whole image. Multiple channels of information
are simultaneously collected (the multi-channel advantage).
Imaging Vs. Mapping
As opposed to image build-up by point-by-point FTIR mapping, FTIR imaging is used to
describe the collection of a chemical profile of the sample area in a single experiment.
The major instrumental difference between a mapping and an imaging instrument is the
incorporation of a Focal Plane Array detector at the end of the optical train in the
microscope. Average chemical information (i.e. the infrared spectrum) of a specific
spatial area on the sample can be uniquely correlated to the output of a pixel in the FPA.
Hence, no apertures are required to limit the sample area examined. Moreover, the
sample does not need to be moved as a given field of view is imaged in a single
collection experiment. It may be immediately seen that the collection time is decreased
by a factor of n2, where n is the number of spatial resolution elements in one direction of
a square sample area imaged (n is the number of steps in a mapping experiment
considered equivalent to a pixel in the imaging experiment). Moreover, the practical
spatial resolution limit in the mapping technique is close to 10 microns. For imaging, the
resolution is essentially wavelength limited. Hence, the imaging technique has allowed
for the collection of images in faster time with higher resolution. Some phenomena have
also been examined in real time using FTIR imaging. More details can be found in the
applications section.
FTIR Imaging Spectrometry
Instrumentation: The Bio-Rad Stingray imaging spectrometer was used to acquire
infrared images. It consists of a step scan interferometer bench (FTS 6000) coupled to a
Focal Plane Array (FPA) detector equipped microscope, UMA-500. The microscope
detector is a Mercury Cadmium Telluride (MCT) array of 64 x 64 elements imaging an
average spatial area of 500 micron x 500 micron in a single experiment. A ZnSe lens is
used to focus the sample area onto the FPA. To improve signal to noise characteristics,
the instrument is equipped with a Germanium long pass filter and KRS-5 plate, which
acts as a diffuser. The long pass filter serves to prevent detector pixel saturation and
removes background noise outside the bandwidth of interest. The lightly sanded KRS-5
plate is placed in the beam path before the condenser to further improve spatial
homogeneity in the camera field of view and prevent the detector elements in the center
of the array from saturating. The diffuser was not required for the sample single beams as
the dispersed liquid droplets scattered light and reduced the maximum flux.
Image Collection Parameters: Stepping rates of 10 Hz, 5 Hz, 2.5 Hz and 1 Hz have
generally been used with co-added frames being 3, 20, 80, and 200 respectively at a rate
of frames per second for each image. The integration time per frame was set to 0.0931
ms. A stepping rate of 25 Hz did not give reliable performance of the spectrometer and
co-adding more than 3 frames at 10 Hz lead to the same problem. A total of 4096 single
beam images were obtained in each experiment after fast fourier transforming the
interferograms collected at an undersampling ratio (UDR) of 4 and spectral resolutions of
8 cm-1 or 4 cm-1. These are typical collection parameters for our samples on our
instrument and will, in general, vary with the samples and results desired on others.
Data Processing and Noise Reduction
Data Processing: One of the major differences between IR imaging and conventional
FTIR microspectroscopy is the large amount of data generated in a single imaging
experiment. 4096 (for a 64x64 array) to 65536 (for a 256x256 array) spectra acquired in a
single experiment are common numbers. Special data handling techniques and software
are required for data analysis. Image acquisition and preliminary processing is carried out
using the instrumental software, Win-IR Pro.
A hyperspectral imaging software package, the Environment for Visualizing Images
(ENVI), was used for further processing the image cube. Custom routines for
mathematical manipulations, baseline correction, and noise estimation were written in
IDL and incorporated into ENVI. Morphological analyses were carried out using a
commercial image analysis software package, Sigma Scan Pro. All spectral image
computations are performed on a relatively fast Windows NT desktop computer (450
MHz Pentium III processor/ 128 MB RAM) optimized for running ENVI. Image analysis
using Sigma Scan Pro was carried out on a slower computer (133 MHz/32 MB RAM)
running Windows 95.
Improving Image quality
Introduction
The Signal to Noise Ratio (SNR) achieved in FTIR Imaging data varies considerably
with the data collection parameters. In general, the SNR levels for spectra collected using
the step scan mode are lower compared to spectra collected in the rapid scan mode.
Moreover, the FPA has its own unique sources of noise. A number of factors affecting
the SNR of spectra obtained in the imaging experiment are discussed in the section
below.
Sources of Noise
The sources of noise are varied and may be broadly categorized based on their origin.
The noise sources are
1. Detector Noise
2. Spectrometer Noise: Sources of noise in a step scan FTIR spectrometer have been a
source of discussion in the literature. An excellent article has been published describing
the commonly encountered sources of noise (Manning C.J., and Griffiths P.R., Appl.
Spectrosc., 1997, 51, 1092).
2. The sample: The sample introduces it's own means of lowering the SNR in many
cases. Since the very reason to use FTIR imaging is to examine multiphase structures,
there is a natural existence of variations in refractive index and transparency across the
spatial area examined. The sample is the most difficult noise source to characterize.
Noise Reduction by Hardware Improvement
Clearly, one route to lower noise data is to have lower noise FPA detectors, i.e. detectors
with lower noise electronics, more efficient photon response, and deeper wells etc.
However, expecting such dramatic improvements every small interval of time is not a
realistic situation. A situation where the detector may need to be replaced is when pixels
start "dropping off". Actually, delamination of the IR sensitive material from the
underlying array detector takes place due to the failure of bump bonds between the two.
This leads to non-responsive pixels showing up as black pixels during data collection. In
our experience, the array remains stable for months with the number of such pixels being
less than a fraction of a percentage point. Once failure sets in, the FPA is still in a
satisfactory state with less than a few percent of failed pixels for many months thereafter.
Depending on the tolerance levels of the experiment, catastrophic failure (say greater
than 10% failed pixels) may not occur till months after the first signs of failure. Oddly,
the pixels start to fail from the outside corners first!
Noise Reduction by Collection Techniques
The simplest technique in this category is to increase the number of frames co-added.
However, the benefits of this technique, especially when considering increase in
collection time, are limited. A typical curve showing the SNR as a function of holding
time at each mirror step is shown in the figure below.
The figures next to the data points show the number of coadded frames. Clearly, coadding more than 80 frames has little impact on the SNR. In most cases, the optimum
number of frames to be co-added are 20. For a spectral resolution of 8cm-1 and a UDR
setting of 4, a collection time of approximately 3.5 minutes is achieved for 20 co-added
frames.
A collection technique was suggested by Snively and Koenig to increase the image SNR.
This was similar to classical co-addition in that absorbance image files were co-added.
The improvement is the SNR was found to scale as (n)1/2
where n are the number of absorbance image files co-added. Clearly, the time required to
attain higher SNR images exceeds the benefits by a factor of (n)1/2. An example of the
effectiveness of this method can be seen in the figure below
When faster events are required to be monitored, such an approach is not very effective
as it is time-consuming. Another approach to obtain less noisy data is to co-add pixels
that are assumed to have the same true values based on physical arguments. e.g. a column
of pixels in the contact method experiments should have the same spectral features. The
SNR improvement again scales as (n)1/2 where n are the number of pixels co-added.
Post-Collection Noise Reduction
This is achieved by using mathematical procedures to eliminate noise. One such
procedure is employed by using the Minimum Noise Fraction transform. Details on the
application of this transform to reduce noise in FTIR spectroscopic images can be found
here.
Some applications
The coupling of an FTIR spectrometer with an FPA was pioneered by researchers at the
Laboratory of Chemical Physics, NIDDK at NIH. Dr. E. Neil Lewis and Dr. I. W. Levin
from the NIH, in collaboration with Researchers from the Miami Valley Laboratories at
Procter and Gamble: Drs. C. Marcott, R.C. Reeder, A.E. Dowery, G.M. Story and Dr. P.J.
Treado from ChemIcon Inc. published a groundbreaking paper using this technique
(Anal. Chem., 67, 3377 (1995) ). Researchers and developers at Bio-Rad have developed
the concept into a commercial product by combining a state of the art spectrometer
bench, the FTS 6000 featuring the 896 interferometer design, with their UMA 500
microscope accessory. Another organization, Spectral Dimensions, has started to
manufacture FTIR imaging systems.
The spectroscopy laboratories at CWRU, directed by Prof. Koenig, were among the first
ones to obtain this instrument and have applied it to study polymeric systems over the last
2+ years. All the work reported on these pages is a product of these laboratories. There
are other active groups with numerous publications on the applications of this type of
imaging spectrometry. The group at NIH (headed by Dr. Levin) concentrates on the
biological aspects of the applications of FTIR imaging. The group at P&G (notably Dr.
C. Marcott) have examined polymer laminate films, wheat grains and biological
specimens. Other researchers imaging biological specimens include Dr. Treado of Chem
Icon Inc. Chris Snively, who did his doctoral work in our group, is now looking at the
applications of FTIR imaging to the field of combinatorial chemistry at Purdue. Dr.
Norman Wright and co-workers at Bio-Rad are actively involved in the dissemination of
Stingray technology : in the design as well as some application aspects. The work of
these researchers may be found in many conference presentations and articles (a small list
is available here). A search on these researchers using a good database over the last 5-6
years should reveal a large majority of the published/presented literature on FTIR
imaging spectroscopy using an FPA.
Only some representative examples from my Ph.D. thesis work are reported on these
pages. Please refer to the actual publications or request a copy from us for complete
details. Please feel free to send email suggesting links to imaging sites and other work. If
you would like to contribute some text or images to the site, please send it to me via
email.
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