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Fourier-Transform Mid-Infrared Focal Plane Array Imaging ...
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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. [email protected] Nematode Worms 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. Heterorhabditis heliothidis 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 3000 2900 1800 1600 1400 1200 1000 -0.008 -0.006 -0.004 -0.002 0.000 0.002 0.004 B Absorbance 1 2 3 4 5 A Wavenumber (cm -1 ) A Fibre Model 0.0 0.1 0.2 0.3 0.4 0.5 3250 3000 2750 2500 2250 2000 1750 1500 1250 1000 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 b - after correction Centre Top Edge Bottom Edge Reference a - before correction Absorbance Wavenumber (cm -1 ) Centre Top Edge Bottom Edge Reference Comparison Between Species Experimental Procedures The nematodes, and , 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 , 4 cm 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 ( . 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 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. S. feltiae H. heliothidis -1 -1 -1 P. Bassan et al, Analyst (2010) 135: 268-277) 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 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. A previous study by Ami (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 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 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 individuals, with a number of clusters observed associated specifically to this species, but that few clusters are shared between individuals from different species. et al. H. heliothidis S. feltiae H. heliothidis Visual images (left) and HCA analysis (below) for seven different nematodes, two (labelled Sf) and five (labelled Hh) individuals. S. feltiae H. Heliothidis 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. B.L. acknowledges funds received from the Austrian Research Agency within the K-Project Process Analytical Chemistry
Transcript of Fourier-Transform Mid-Infrared Focal Plane Array Imaging ...
Fourier-Transform Mid-Infrared Focal PlaneArray Imaging of a Complex
Multicellular OrganismAlison J. Hobro and Bernhard Lendl
Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164-AC, A-1060 Vienna, Austria. [email protected]
Nematode Worms
Nematodes are multicellular organisms with defined digestive, nervous, reproductive andlocomotive systems analogous to those found in higher organisms. However, their overallphysical shape and characteristics are considerably more complex compared to that of celllayers 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.
Heterorhabditis heliothidis
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
3000 2900 1800 1600 1400 1200 1000
-0.008
-0.006
-0.004
-0.002
0.000
0.002
0.004 BAb
so
rba
nce
1
2
3
4
5
A
Wavenumber (cm-1)
A Fibre Model
0.0
0.1
0.2
0.3
0.4
0.5
3250 3000 2750 2500 2250 2000 1750 1500 1250 1000
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
b - after correction
Centre
Top Edge
Bottom Edge
Reference
a - before correction
Ab
so
rba
nce
Wavenumber (cm-1)
Centre
Top Edge
Bottom Edge
Reference
Comparison Between Species
Experimental ProceduresThe nematodes, and , was purchased as dried media from Biohelp GmbH (Austria). These were rinsed several times, re-suspended in waterand allowed to stand for approximately 20 minutes before opening carefully and spotting small volumes onto a mid-IR transparent zinc selenide plate (ZnSe). As thewater evaporates the nematodes adhere to the ZnSe slide and cannot move during the IR measurements. The polypropylene fibres were obtained from AndreasBartl (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 , 4 cm 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 ornematode images a background spectrum was collected from the ZnSe plate outside of the areas with the fibre or nematode suspension. The IR referencespectrum of the polypropylene fibre was also collected using the Hyperion 3000 IR microscope, this time operating in single point mode using a mercury cadmiumtelluride (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 imagebefore applying the correction algorithm and subsequent image analysis.
Spectra were corrected for Mie scattering effects using a correction algorithm operating in Matlab ( . The correctionsoftware used has two options, one for Mie scattering correction and one for resonant Mie scattering correction. Comparison of the performance of these twoalgorithms when applied to the polypropylene fibre image suggested that the Mie scattering correction was more effective and so this was used for the nematodeimage corrections. The Mie scattering correction algorithm was applied using a Matrigel reference spectrum, 8 principle components, the size of the scatteringparticle 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 thenematode spectra from other spectra e.g. originating from the ZnSe slide or cellular debris. The IR spectra in the image were also transformed into secondderivative spectra (using an 11 point smoothing function) before cluster analysis. Hierarchical Cluster Analysis (HCA) was performed CytoSpec limiting the spectralregions of interest to 1000-1750 and 2830-3000 cm using the D-value distance method. Ward's Algorithm was used to cluster the data and the appropriatenumber of clusters defined as when the average spectrum for each of the clusters are sufficiently different but that the standard deviation of the spectra containedwithin each cluster is low. The multiple worm image was constructed by amalgamating individual images into one matrix in Matlab and the combined imageprocessed in the same manner as the individual worm, with an additional step of normalisation of the derivative spectra prior to HCA.
S. feltiae H. heliothidis
-1 -1
-1
P. Bassan et al, Analyst (2010) 135: 268-277)
In order to test the effectiveness ofcorrection algorithms for removingscattering based artefacts a polypropylenefibre was imaged. Before correction (a),individual spectra taken from different areasshow clear baseline oscillations. However,there is no evidence of a sharp negative'dispersion artefact', nor is there anyevidence of shifts in band position betweenthe image reference spectra as would bepredicted if these distortions were due toresonant Mie scattering .
Upon Mie scattering correction (b), themajority of the longer frequency oscillationsin the baseline are removed, along with thebaseline offset observed in the spectraclosest to the fibre edge. The variationbetween spectra taken from the sameregion is significantly reduced and, althoughsome differences are still apparent betweenspectra taken from the centre and the edgeof the fibre, these differences are small inthe regions of interest
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 topof each other, rather than the spectra from each pixel representing one cell thickness wherespectral 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 withorgans (pharyngeal muscles, stomatointestinal muscles, the anal sphincter or gonadal andsex-specific muscles).Grey: Also protein rich. Possibly associated with associated with the somatic muscle foundalong the length of the body wallGreen: Collagen rich. Associated with the cuticle.
A previous study by Ami (FEBS letters (2004) 576:297-300) used single point IRspectra to analyse nematode worms and found that the spectral signature of the pharynxregion was sufficient to differentiate between species, whereas the intestine and tail regionsdid not give rise to such reproducible spectra.
In all cases, for both species, the spectra of the pharynx regions are separated into a numberof clusters. All the individuals contain the clusters depicted in light blue andwhite, 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 thespectral signatures contained in these clusters are unaffected by the health of the nematode.In contrast individuals show little similarity to each other. These HCA results suggestthat the IR spectra from the pharynx region exhibit a high degree of similarity between
individuals, with a number of clusters observed associated specifically to thisspecies, but that few clusters are shared between individuals from different species.
et al.
H. heliothidis
S. feltiaeH.
heliothidis
Visual images (left) and HCAanalysis (below) for sevendifferent nematodes, two
(labelled Sf) and five(labelled Hh)
individuals.
S.feltiae H.Heliothidis
Conclusions�
�
�
�
�
Although a nematode is a thicker tissue with an essentially cylindrical shape and morecomplex multicellular composition than has previously been studied by IR imaging, suchanalysis has been shown here to be possible.
Initial tests using a uniform polypropylene fibre showed that distortions in the spectralbaseline can be observed, especially towards the edges of the fibre but application of analgorithm developed for Mie scattering correction significantly reduced these distortions.
Although clustering analysis only finds small differences in spectral profile between differentareas of the nematode these differences can be attributed to the digestive tract, cuticle, anddifferent protein constituents in the body cavity.
HCA clusters can be associated specifically to a particular species, but that few clusters areshared between individuals from different species.
IR imaging of nematodes has the potential to differentiate between species, whilst alsoproviding information on the distribution of biomarkers within each individual.
B.L. acknowledges funds received from the Austrian Research Agency within the K-Project Process Analytical Chemistry
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