Fourier-Transform Mid-Infrared Focal Plane
Array Imaging of a Complex
Multicellular Organism
Alison J. Hobro and Bernhard Lendl
Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164-AC, A-1060 Vienna, Austria.
A Fibre Model
Nematodes are multicellular organisms with defined digestive, nervous, reproductive and
locomotive systems analogous to those found in higher organisms. However, their overall
physical shape and characteristics are considerably more complex compared to that of cell
layers or tissue sections. From the perspective of IR spectroscopy a nematode is thick,
consisting of many layers of cells in the centre, making spectral interpretation complex.
In order to test the effectiveness of
correction algorithms for removing
scattering based artefacts a polypropylene
fibre was imaged. Before correction (a),
individual spectra taken from different areas
show clear baseline oscillations. However,
there is no evidence of a sharp negative
'dispersion artefact', nor is there any
evidence of shifts in band position between
the image reference spectra as would be
predicted if these distortions were due to
resonant Mie scattering .
Upon Mie scattering correction (b), the
majority of the longer frequency oscillations
in the baseline are removed, along with the
baseline offset observed in the spectra
closest to the fibre edge. The variation
between spectra taken from the same
region is significantly reduced and, although
some differences are still apparent between
spectra taken from the centre and the edge
of the fibre, these differences are small in
the regions of interest
Absorbance
0.004
a - before correction
Centre
Top Edge
Bottom Edge
Reference
b - after correction
Centre
Top Edge
Bottom Edge
Reference
0.4
0.3
0.2
0.1
0.0
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
3250
3000
2750
2500
2250
2000
1750
1500
1250
1000
-1
Wavenumber (cm )
Comparison Between Species
Heterorhabditis heliothidis
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0.5
Absorbance
Nematode Worms
[email protected]
A
Visual images (left) and HCA
analysis (below) for seven
different nematodes, two S.
feltiae (labelled Sf) and five H.
Heliothidis (labelled Hh)
individuals.
1
2
3
4
5
B
0.002
0.000
-0.002
-0.004
-0.006
-0.008
3000 2900 1800
1600
1400
1200
1000
-1
Wavenumber (cm )
The similarity of the different clusters (absorbance spectra (a) and 2nd derivative spectra (b))
could be anticipated from the structure of the nematode where, as a relatively thick object
(approximately 20 μm), each pixel in the IR image is an average of several cells stacked on top
of each other, rather than the spectra from each pixel representing one cell thickness where
spectral differences would be more marked.
Dark blue: Lipid rich. Associated with the digestive tract.
Light blue: Protein rich. Possibly associated with non-striated muscle cells associated with
organs (pharyngeal muscles, stomatointestinal muscles, the anal sphincter or gonadal and
sex-specific muscles).
Grey: Also protein rich. Possibly associated with associated with the somatic muscle found
along the length of the body wall
Green: Collagen rich. Associated with the cuticle.
Conclusions
? Although a nematode is a thicker tissue with an essentially cylindrical shape and more
complex multicellular composition than has previously been studied by IR imaging, such
analysis has been shown here to be possible.
? Initial tests using a uniform polypropylene fibre showed that distortions in the spectral
baseline can be observed, especially towards the edges of the fibre but application of an
algorithm developed for Mie scattering correction significantly reduced these distortions.
? Although clustering analysis only finds small differences in spectral profile between different
areas of the nematode these differences can be attributed to the digestive tract, cuticle, and
different protein constituents in the body cavity.
? HCA clusters can be associated specifically to a particular species, but that few clusters are
shared between individuals from different species.
? IR imaging of nematodes has the potential to differentiate between species, whilst also
providing information on the distribution of biomarkers within each individual.
A previous study by Ami et al. (FEBS letters (2004) 576:297-300) used single point IR
spectra to analyse nematode worms and found that the spectral signature of the pharynx
region was sufficient to differentiate between species, whereas the intestine and tail regions
did not give rise to such reproducible spectra.
In all cases, for both species, the spectra of the pharynx regions are separated into a number
of clusters. All the H. heliothidis individuals contain the clusters depicted in light blue and
white, while most also contain the light green, dark green, brown and salmon pink clusters.
This is also the case for the damaged individual (noted by the asterisk) suggesting that the
spectral signatures contained in these clusters are unaffected by the health of the nematode.
In contrast S. feltiae individuals show little similarity to each other. These HCA results suggest
that the IR spectra from the pharynx region exhibit a high degree of similarity between H.
heliothidis individuals, with a number of clusters observed associated specifically to this
species, but that few clusters are shared between individuals from different species.
Experimental Procedures
The nematodes, S. feltiae and H. heliothidis, was purchased as dried media from Biohelp GmbH (Austria). These were rinsed several times, re-suspended in water
and allowed to stand for approximately 20 minutes before opening carefully and spotting small volumes onto a mid-IR transparent zinc selenide plate (ZnSe). As the
water evaporates the nematodes adhere to the ZnSe slide and cannot move during the IR measurements. The polypropylene fibres were obtained from Andreas
Bartl (Vienna University of Technology) and were prepared following the same procedure.
IR images of the polypropylene and the nematode were measured using a Bruker Hyperion 3000 IR microscope, attached to a Tensor 37 FT-IR spectrometer,
operating in focal plane array (FPA) imaging mode. The image spectra were collected in the spectral range 1000-3300 cm-1, 4 cm-1 resolution and 128 scans,
using a x15 objective and an FPA detector with 64 x 64 pixels. Due to the size of the nematodes the images presented here are a composite of 2 x 3 FPA images,
resulting in a final measured area of 350 x 525 μm. All image spectra were collected as absorbance spectra in transmission mode. Prior to recording the fibre or
nematode images a background spectrum was collected from the ZnSe plate outside of the areas with the fibre or nematode suspension. The IR reference
spectrum of the polypropylene fibre was also collected using the Hyperion 3000 IR microscope, this time operating in single point mode using a mercury cadmium
telluride (MCT) detector. Due to the size of some of the nematode IR images the pixels were binned (2x2 pixels) in order to reduce the number of pixels in the image
before applying the correction algorithm and subsequent image analysis.
Spectra were corrected for Mie scattering effects using a correction algorithm operating in Matlab (P. Bassan et al, Analyst (2010) 135: 268-277). The correction
software used has two options, one for Mie scattering correction and one for resonant Mie scattering correction. Comparison of the performance of these two
algorithms when applied to the polypropylene fibre image suggested that the Mie scattering correction was more effective and so this was used for the nematode
image corrections. The Mie scattering correction algorithm was applied using a Matrigel reference spectrum, 8 principle components, the size of the scattering
particle set to 2-18 μm and the refractive index set to 1.1-1.5.
All IR image reconstruction, display and cluster analyses were performed in CytoSpec (www.cytospec.com). The region of interest tool was used to isolate the
nematode spectra from other spectra e.g. originating from the ZnSe slide or cellular debris. The IR spectra in the image were also transformed into second
derivative spectra (using an 11 point smoothing function) before cluster analysis. Hierarchical Cluster Analysis (HCA) was performed CytoSpec limiting the spectral
regions of interest to 1000-1750 and 2830-3000 cm-1 using the D-value distance method. Ward's Algorithm was used to cluster the data and the appropriate
number of clusters defined as when the average spectrum for each of the clusters are sufficiently different but that the standard deviation of the spectra contained
within each cluster is low. The multiple worm image was constructed by amalgamating individual images into one matrix in Matlab and the combined image
processed in the same manner as the individual worm, with an additional step of normalisation of the derivative spectra prior to HCA.
B.L. acknowledges funds received from the Austrian Research Agency within the K-Project Process Analytical Chemistry