Journal of Molecular Structure 933 (2009) 104–111

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Journal of Molecular Structure

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FTIR microspectroscopic imaging as a new tool to distinguish chemicalcomposition of mouse blastocyst

Kanjana Thumanu a,*, Waraporn Tanthanuch a, Chanchao Lorthongpanich b, Philip Heraud c,Rangsun Parnpai b

a Synchrotron Light Research Institute (Public Organization), 111 University Avenue, Muang, Nakhon Ratchasima 30000, Thailandb Embryo Technology and Stem Cell Research Center, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasiam, Thailandc Monash Immunology and Stem Cell Laboratories, and The Centre for Biospectroscopy, School of Chemistry, Monash University, Clayton, Victoria 3800, Australia

a r t i c l e i n f o a b s t r a c t

Article history:Received 26 February 2009Received in revised form 5 June 2009Accepted 5 June 2009Available online 9 June 2009

Keywords:Focal plane array (FPA) imagingSynchrotron-Infrared (SR-IR) mappingBlastocystPrinciple Component Analysis (PCA)Hierachical Cluster Analysis (HCA)

0022-2860/$ - see front matter Crown Copyright � 2doi:10.1016/j.molstruc.2009.06.003

* Corresponding author. Tel.: +66 44217040; fax: +E-mail address: kthumanu@slri.or.th (K. Thumanu

Synchrotron-Infrared (SR-IR) mapping and Focal plane array (FPA) imaging have been applied for dis-crimination of the three biochemical components of the mouse blastocyst. The mouse blastocyst consistsof two clusters of cells known as the inner cell mass (ICM) formed within the blastocoel cavity and thethin layer of outer cells called the trophectoderm. Using Hierachical Cluster Analysis (HCA) and PrincipleComponent Analysis (PCA), it can be shown that the composition and distribution of biochemical compo-nents within the blastocyst show differences in the protein secondary structure and the lipid content. It isworth noting that the secondary structure of the outer layer cells indicates more distinctive b-type sec-ondary structure. The blastocoel cavity was observed to be predominantly a-helix. Significantly, the ICMregion showed the predominant high absorption intensities of lipid content (CH2, CH3 symmetric andasymmetric stretching around 3000–2800 cm�1). The results show agreement between both SR-IR map-ping and FPA-IR imaging. We propose that the biochemical difference within the blastocyst, especiallythe high lipid content in the ICM region, could be involved in the process of lipid signaling during pre-implantation. The use of both techniques is shown to be significant approach for revealing the biochem-ical components within the blastocyst.

Crown Copyright � 2009 Published by Elsevier B.V. All rights reserved.

1. Introduction

Traditionally, the gold standard for analysis of biological sam-ples is based on histopathological assessment of the sample, re-lated to a number of specific immunological staining procedures.The standard technique is used to determine cell types using spe-cific antibody labels which recognize and bind specific antigens.Although, these methods are routinely used, they require accuratepreparation, which are time-consuming and expensive. Fouriertransform infrared (FTIR) microspectroscopy has been introducedas a new tool for chemical analysis of biological and biomedicalsamples [1–4]. This new technique is non-destructive and lessexpensive when compared with the immunostaining method fordetection of specific proteins [5–7]. This alternative method is ra-pid as it allows the examination of the expression of different bio-markers simultaneously in a single absorption measurement. TheFTIR spectrum of biological samples can provide detailed spectralinformation on cellular components such as nucleic acid, protein,lipid in the mid-IR spectral region between 4000 and 700 cm�1

known as ‘‘fingerprint region” of the spectral domain [8,9].

009 Published by Elsevier B.V. All

66 44217047.).

A more promising tool for a new generation of IR microspec-troscopy is the Focal plane array detector (FPA) [2,10–14]. FTIRimaging based on FPA permits, rapid examination, of spatially re-solved chemical information, from large sample areas, in a rela-tively short time, dramatically improving data acquisitions rate.Chemical imaging of the different structural components of biolog-ical samples can be imaged based on the vibrational signature ofthe sample components.

Synchrotron-Infrared (SR-IR) microspectroscopy has been intro-duced to identify molecular constituents in biological samples. With100–1000 times brightness advantage of the SR source over the glo-bar IR source, SR-IR microspectroscopy is well-suited for analyzingsamples which require the characterization of a very small area tomaximize the advantage of the SR-IR source [15–18]. The highbrightness of the synchrotron source allows small regions to bedetected with high signal to noise ratio and highly spatial resolution.However, SR-IR measurement has a main barrier as the fact that datacan be acquired point by point spectral acquisition using synchro-tron based mapping with a single point detector element. Therefore,the collection of individual spectra over large areas of sample is verytime-consuming (takes hours).

During the process of embryogenesis by which the embryo isformed and develops, the fertilized eggs grow into a ball of cells

rights reserved.

K. Thumanu et al. / Journal of Molecular Structure 933 (2009) 104–111 105

known as blastocyst. The blastocyst consists of the two primarycells; inner cell mass (ICM) and trophoblast (TE) cells. ICM areformed within the blastocoel (called blastocyst cavity) which isthe fluid-filled center region of a blastocyst. The ICM develops intoa fetus and a thin layer of outer cells called TE give rise to the pla-centa [19–21]. ICM is an important source of embryonic stem (ES)cells which could be induced and differentiated to form all celltypes of the body. Moreover, the ES cells could also provide animportant tool for medical examinations such as drug testing, em-bryo development and cell differentiation [22–24].

There are some literature reviews which attempt to understandthe mechanisms of mouse embryo implantation [25–29]. It hasbeen reported that mouse blastocyst releases a lipid anadanamide(AEA) that activates fatty acid amide hydrolase (FAAH) which is themetabolic gatekeeper for AEA signaling.

Normally, to understand the mechanisms responsible for em-bryo development and implantation events in mice, the standardtechniques using molecular, biochemical and physiological ap-proached were applied to study the expression of N-acylphospha-tidylethanolamine-selective phosphate lipase D (NAPE-PLD),FAAH, CB1 receptor in the mouse oviduct and pre-implantationembryo [28,31].

Among the spectroscopic methods, FTIR is one of the vibrationalspectroscopy techniques that can reveal the nature of a biologicalsample and can produce spectral maps of compositional and struc-tural changes without marker on the molecular level. Recently, FTIRwith FPA detectors have been used to identify the differentiation

Fig. 1. Molecular functional group image of the mouse blastocyst analyzed by FPA-IR Imcovered an area of 340 � 340 lm. The IR image (64 � 64 pixels) was generated with a 3used to indicate absorbance, with the highest absorbance (red color) and the lowest abgroups map obtained from original spectra and 2nd derivative and normalized at absorbcorresponding to the pixel at the cross-hair in the visible image at position 1–3. (For interof this article.)

state of individual human mesenchymal stem cells with and with-out initiation of a differentiation process [35]. Also, conventionalFTIR microspectroscopy has also been used to monitor spontaneousdifferentiation of murine ES cells [36]. In this study, we apply bothtechniques to distinguish structural information by creating a spec-troscopic database which contains statistical information from FTIRspectra of the mouse blastocyst. This work helps us to reveal differ-ent biochemical components of mouse blastocyst especially in theICM region which could be involved in the regulation of the cell sig-nal during the process of blastocyst activation. Coupled with PCAand HCA, we demonstrated that FPA-IR imaging and SR-IR mappingenabled us to identify spectral changes and distinguish three bio-chemical components of the mouse blastocyst.

2. Materials and methods

2.1. Animals

Female outbred and inbred stock mice (ICR and C57BL/6 strain;4–6 weeks old) were superovulated with 10 IU pregnant mare ser-um gonadotropin (Sigma). This was followed by 10 IU human cho-rionic gonadotropin (hCG, Sigma; i.p.) 48 h later, and then bynatural mating with the same strain of male mice. Two-cell (2C)embryos were collected at 43–45 h after the hCG injection fromthe oviducts and then cultured in 20 ml drops of CZB media [37]under mineral oil with 5% CO2 in air at 37 �C until developmentto the blastocyst.

aging. A mosaic of 4 FTIR images was sequentially recoded in a 2 � 2 pattern which2 � 32 pixel FPA detector (128 scans, 6 cm�1 resolution). The color-coded has beensorbance (blue color). Visual image and FTIR Overview image based on functionalance value (a) 2920 cm�1, (b) 1635 cm�1and (c) 1653 cm�1. Representative spectrapretation of color mentioned in this figure, the reader is referred to the web version

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2.2. Sample preparation for FTIR microspectroscopy

The mouse blastocysts (ICR and C57BL/6 strain; outbred andinbred strains, respectively) were supplied by Embryo Technologyand Stem Cell Research Center and School of Biotechnology, Insti-tute of Agricultural Technology, Suranaree University. They werewashed three times with 0.9% NaCl and then deposited onto in-dium tin oxide-coated, silver-doped glass slides (MirrIR, Tienta Sci-ences, Ohio, USA) and vacuum dried for 24 h in a desicator prior toSR-IR mapping and FPA-IR imaging.

2.3. Methods

2.3.1. Measurement using a FPA detectorThe experiments were performed at the IRPC Public company

limited, Rayong, Thailand, utilizing a Vertex 70 FTIR spectrometerconnected to an IR microscope (Hyperion 3000). The microscopeis equipped with a 32 � 32 element MCT, FPA detector, which al-lows simultaneously acquisition of spectral data with a 15� Casse-grain objective. FTIR samples were recorded in reflection mode,6 cm�1 spectral resolution, 128 scans per spectrum. Each of thefour sections used to construct 2 � 2 FTIR image mosaic fromFPA recording which covered an area of 340 � 340 lm. Absorbancespectra were acquired in the spectral range of 4000–700 cm�1.

2.3.2. Synchrotron FTIR microspectroscopy measurementSR-IR measurement was obtained at IR microspectroscopy

beamline, the Australian synchrotron, Melbourne, Australia. Spec-

Fig. 2. FPA-IR imaging of the mouse blastocyst. (a) 2D cluster image using five clusters bathe cluster areas. A 32 � 32 pixel MCT FPA detector was used to image the cell, where tscans, 6 cm�1 resolution). (b) Representative original average spectra of the spectra clust2, 1 and 5), the blastocole cavity (cluster 4). (c) Average cluster spectra from a six clureferences to color in this figure legend, the reader is referred to the web version of thi

tra were recorded with a (Vertex 80v) IR spectrometer coupledwith an IR microscope (Hyperion 2000) and MCT detector cooledwith liquid nitrogen with measurement range of 4000–600 cm�1.The high brilliance of SR-IR allowed an aperture of 5 � 5 lmwhich achieved a high S/N ration and high spatial resolution.Spectral collection was made in reflection mode at 6 cm�1

resolution, 32 scans were co-added and converted to absor-bance using OPUS 5.0 software. (Bruker Optics Ltd, Ettlingen,Germany).

2.3.3. Data processing for image analysisIR imaging of mouse blastocyst was constructed and analyzed

using Cytospec 1.3.4 (Cytospec Inc., NY, USA). Preprocessing ofthe data was required to remove spectra showing signs of miescattering, dispersion artifacts, sloping baselines or poor qualityspectra. To remove poor quality spectra, the thickness qualitytest was used to eliminate spectra of samples which were toothin or thick. Then, the spectra were converted to the 2nd deriv-ative, using 13 smoothing points, and vector normalized to nor-malize for the effects of differing sample thickness. The imageconstruction can be processed using univariate mode which gen-erate based on peak intensity, peak area or peak ratio to yield,commonly referred to as chemical maps or function group maps.HCA was performed to distinguish the different biochemicalcomponents of the mouse blastocyst over spectral ranges of3000–2800 cm�1 and 1800–900 cm�1. The individual 2D clustermaps were recorded as an image file with a unique color to as-sign each cluster.

sed on the range 3000–2800 cm�1 and 1800–900 cm�1. Color-coded corresponds tohe resulting field of view on the FPA was 175 � 175 lm with 5.5 lm per pixel (128er as obtained by HCA (5 clusters). The ICM (cluster 2), the outer layers cells (clusterster analyses after 2nd derivative and vector normalize. (For interpretation of thes paper.)

K. Thumanu et al. / Journal of Molecular Structure 933 (2009) 104–111 107

2.3.4. Multivariate data analysisIndividual spectra from each cluster were analyzed using PCA to

distinguish different biochemical components of the blastocyst.The spectra were processed using 2nd derivative and vector nor-malized by the Savitzky-Golay method (3rd polynomial, 9 smooth-ing points) using the Unscrambler 9.7 software. (CAMO, Norway).

3. Results and discussion

The IR microspectroscopy technique provides a spectral signa-ture of the chemical components within the mouse blastocyst.Spectra acquired from both FPA-IR imaging and SR-IR mapping dis-play the distinctive IR spectra signature of specific molecular struc-tures such as protein and lipid [8].

3.1. Imaging molecular chemistry of the mouse blastocyst

Fig. 1 shows color maps of functional groups of outbred mouseblastocyst with an area of 175 � 175 lm analyzed by FPA-imaging.The visual image of the mouse blastocyst, clearly shows that theICM of mouse blastocyst was located in the middle of the blasto-cyst. The chemical image in false-color yields a representation ofthe chemical component intensities and 2nd derivative spectraafter vector normalization for differences in sample thickness.Absorbance intensities of FTIR spectra maps are proportional tocolor changes: blue (lowest) < green < yellow < red (the highestintensity). The molecular chemistry of functional groups was im-aged at 2920 cm�1 (Asymmetric stretching of CH2 vibration), at1653 cm�1 (amide I C@O of a-helix) and 1635 cm�1 (amide I

Fig. 3. Molecular functional group image of the mouse blastocyst analyzed by SR-IR mobtained from absorbance value (a) 2920 cm�1, (b) 1653 cm�1and (c) 1635 cm�1after 2ndthe cross-hair in the visible image at position 1–3. Spectrum pixel size 5 � 5 lm. (e) Ve

C@O of b-sheet). This shows the distribution and relative concen-tration of the chemical groups associated with the biochemicalcomponent within the mouse blastocyst. As seen in Fig. 1a, thehigh lipid content centered at 2920 cm�1 was located in the ICMregion of the blastocyst. The outer layer of the cells shows a highb-sheet content (Fig. 1b), while, a high a-helix content was indi-cated in the blastocoel cavity of the blastocyst (Fig. 1c).

The most useful method for direct comparison is HCA whichaims to clarify spectra based on similarity and difference in themacromolecule chemistry of the different cells and tissues[2,14,38]. HCA analysis included average spectra of spectral clus-ters which can be easily obtained and stored for further analysis.In the IR image of mouse blastocyst, we performed multivariatecluster analysis with all spectra.

The results of cluster analysis for mouse blastocyst are dis-played as dendrograms and IR images. (Fig. 2a) Cluster analysiswas performed over the frequency range 3000–2800 cm�1 and1800–900 cm�1 with 2nd derivative data obtained from the origi-nal FTIR spectra. The high correlation of clusters is establishedfor each class related to spectral features of biochemicaldifferences between clusters. The major spectral difference of theinbred mouse blastocyst is the intensity of lipid and protein whichshow distinct bands at 3000–2800 cm�1 and 1700–1600 cm�1,respectively.

Each cluster is displayed in a different color and identifies themajor tissue types in the blastocyst based on differences in macro-molecular chemistry. For example in Fig. 2a, the grey colored clus-ter (cluster 3) represents predominantly ICM formed within theblastocoels cavity which significantly appears in the light blue

apping. Visual image and FTIR Overview image based on functional groups mapderivative and normalized. (d) Representative spectra corresponding to the pixel at

ctor normalized and 2nd derivative of representative spectra at position 1–3.

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colored cluster (cluster 4). While, the red (cluster 2), pink(cluster 5) and blue (cluster 1) colored clusters are slightly differ-ent in spectral information indicated by the outer layer cells.

Fig. 2b and c represent the average spectrum of each cluster,and dendograms showing similarity between spectra. The resultsclearly show that three biochemical components; the ICM, theblastocoel cavity, the outer layer cells can be clearly discriminatedby using HCA. These finding indicate that the overall spectral char-acteristic of mouse blastocyst can be classified by the spectral fea-tures of the amide I band and bands attributable to lipids. Theabsorbance maximum for the amide I band at 1653 cm�1 for theblastocoels cavity indicated high levels of a-helix secondary struc-ture for protein located there, whereas an amide I maximum at1635 cm�1 for the outer layer cells indicated dominance ofb-pleated structure in protein located in these cells. A clear separa-tion of the ICM cluster from all the others can be observed in dend-ograms showing spectral similarity (Fig. 2a), with the averagespectrum for the cluster corroborating the other findings showingthat the ICM to have a high lipid content. The dominant absorptionbands indicative of the high lipid in the ICM are found in the regionof 3000–2800 cm�1 (predominantly asymmetric and symmetricvibration of the CH2 (2920 cm�1) and CH3 (2850 cm�1) groups of

Fig. 4. SR-IR mapping of the mouse blastocyst. (a) 2D cluster image using four clusters bathe cluster areas. The area imaged with the IR microscope was 175 � 175 lm with a squaras obtained by HCA (4 clusters). The ICM (cluster 1), the outer layers cells (cluster 2), the2nd derivative and vector normalize. (For interpretation of the references to color in th

the acyl chains, correlated with the intense lipid ester carbonylband at �1740 cm�1.

Figs. 3 and 4 show chemical maps and HCA of spectra acquiredfrom the mouse blastocysts by SR-IR mapping. Representativespectra (Fig. 3d and e) at position 1, 2 and 3 indicate the absorptionband of the ICM, blastocole cavity and outer layer cells, respec-tively. These results corroborate those described above usingFPA-imaging. For example, SR-IR spectra from the blastocyst cavityshow an intense amide I absorbance at 1658 cm�1 attributed to thedominance of a-helix secondary structure of blastocoel proteins(Fig. 4a; average spectrum for the grey color-coded cluster 3).While, the change in the lipid profile can again also be observedclearly in the ICM region (Fig. 4a; average spectrum for blue col-or-coded cluster 1) showing increase in the intensities of theCH2, CH3 stretching (centered at 2850 and 2920 cm�1) and the es-ter carbonyl band at 1740 cm�1. The higher spatial resolution ofSR-IR method enabled the outer layers of cells of the blastocystbe clearly identified (Fig. 4a). The HCA corroborated the finding de-scribed above for FPA, with average spectrum for the blue color-coded, cluster 2 again indicating a high level b-sheet secondarystructure for outer cell proteins with the amide I absorbance cen-tered at �1635 cm�1.

sed on the range 3000–2800 cm�1 and 1800–900 cm�1. Color-coded corresponds toe aperture size of 5 � 5 lm. (b) Representative average spectra of the spectra clusterblastocole cavity (cluster 3). Average cluster spectra from four cluster analyses afteris figure legend, the reader is referred to the web version of this paper.)

Fig. 5. Score (a) and loading (b) plots from PCA of individual spectra acquired fromSR-IR mapping. The first two PCs are explained by the PC loadings of 43 and 33%,respectively.

K. Thumanu et al. / Journal of Molecular Structure 933 (2009) 104–111 109

The overall spectral characterization has been shown that bothSR-IR mapping and FPA-IR imaging of the mouse blastocyst candiscriminate three biochemical components of the blastocyst. TheFPA-IR measurement has advantage in terms of data acquisitiontime which requires only a few minutes to image the blastocyst.However, the main advantage of SR-IR microspectroscopy is thecapacity of detection at high spatial resolution and high S/N ratiousing very small aperture sizes. For example, we showed that SR-FTIR can discriminate the outer layer cells of the blastocyst moreclearly than when FPA-imaging was employed.

3.2. PCA analysis

The use of multivariate analysis in particular PCA involved inthe process two types of information: clustering and identificationof variables which identified specific wavenumbers, representingvarious molecular groups of the samples.

PCA is a statistical data-reduction method which transforms theoriginal data set of variables into a new set of uncorrected variablesknown as PCs [36,39]. PCA allows us to identify which wavenum-bers in the complex FTIR spectra are significant for the largestspectral variation within mouse blastocyst. This analysis allowsthe relationships between molecular functional groups of variables(lipid, protein) observed on blastocyst samples (ICM, blastocoelcavity, outer layers cells) of FTIR molecular spectra. The output ofthe data analysis can be presented either as two dimensions (twoPCs) or in 3D using three PCs.

To resolve the overlapping of IR spectra, 2nd derivative analysisof the spectra was performed to easily observe small changes in thespectra. The Individual spectra extracted from the region of theICM, fluid, outer cells of the blastocyst were used to demonstratedifferent chemical components of the three region of the blasto-cyst. Using two separate spectral regions (3000–2800 cm�1 and1800–800 cm�1), the PCA analysis can be explained by the mainsource of variation in the fingerprint region of 3000–2800 cm�1

and 1700–1600 cm�1. Scores and loading plots show statisticalanalysis of spectral data involved PCA of a group of individual spec-tra extracted from SR-IR mapping and FPA-IR imaging.

From SR-IR mapping (Fig. 5) results, the most distinguishablecluster represent the spectra from the outer layer cells with allspectra having negative PC1 scores. This can be explained by thepositive loading scores of the PC1 which showed spectral changein the range of 1650–1600 cm�1. Analysis of PC1 loadings explainsdifferent secondary protein structure with predominant b-sheetstructure (1639 cm�1) of the outer layer cells. Loading for PC2showed an opposite correlation between bands in the 3000–2800 cm�1 and 1700–1600 cm�1 spectral ranges, and explainedthe clustering between the blastocoel and ICM spectra observedin the scores plot (Fig. 5a). The high negative loading at1658 cm�1 shown in the loading plot (Fig. 5b) indicated high levelsof a-helical structure in the positively-scored blastocoel spectra,whereas positive loading for band in the 3000–2800 cm�1 for thenegatively-scored ICM spectra indicating high lipid content in theICM region.

3.3. Comparison of average spectra between synchrotron mapping andFPA-IR imaging

From FPA-IR Imaging (Fig. 6a) and SR-IR mapping (Fig. 6b) re-sults, most of spectral changes occur in the protein amide I bands(1700–1600 cm�1) and in the lipid region (3000–2800 cm�1). Theresults show that the highest peak occurs at around 1639 cm�1,associated with the b-sheet secondary structure seen in the spectrafrom outer layer cells and the peak at 1651 cm�1 predominantlyobserved in the spectra of the blastocoel. This result point to ahigher b-sheet and a lower a-helix content of the protein in the

outer layer cells. The peak maximum of the amide I band in the ori-ginal spectra and 2nd derivative spectra of the outer layer clearlyshows a downshift from b-sheet to a-helix secondary structureof the blastocoel. These results indicate that different proteinsare expressed in the mouse blastocyst [32].

Significantly, the band in the region from 3000 to 2800 cm�1

shows high content of lipid in the ICM of mouse blastocyst. Thehigh lipid content is explained by the absorption band of CH2

and CH3 asymmetric stretching at 2958, 2920 and 2850 cm�1.While the secondary structure in the ICM shows both a-helixand additionally a shoulder peak around 1637 cm�1, correspondingto b-sheet. The agreement of the experiment using SR-IR mappingalso gives the same results as compared with FPA-IR imaging.

Due to these facts we can explain three spectral differences ob-served from the biochemical components of the blastocyst. Theexperimental findings of the present study found high lipid contentin the ICM region. To explain these results, we need to discuss theresults in terms of the molecular and physiological function of theAEA lipid signal molecule [27,28,30,32–34].

As reported by Wang et. al. (2005, 2006), AEA lipid signal mol-ecule is at high concentration in the ICM cells of the blastocyst(Fig. 7) [25,27,28,40–42]. There is a negative correlation of bothFAAH and AEA effects on blastocyst survival and implantation.During the pre-implantation event, high levels of AEA lipid signalmolecules which are mostly found in the ICM are hydrolyzed byFAAH. We propose that the low levels of AEA in implantation stage

Fig. 6. Overlay of the 2nd derivative of the average spectra from (a) FPA-IR imaging and (b) SR-IR mapping of mouse blastocyst.

110 K. Thumanu et al. / Journal of Molecular Structure 933 (2009) 104–111

then facilitate implantation. These results conclude that the bipha-sic role of AEA regulates blastocyst activation and implantation.

4. Conclusion

In summary, we have introduced novel SR-based FTIR micro-spectroscopy and FPA-IR imaging to examine the biochemical dif-ference within the blastocyst [2,14,17]. The results showed that thechemical composition distribution such as a-helix (mostly found inthe blastocole cavity) and b-sheet secondary structure (highlyshowed in the outer layer cells) of protein could be imaged. Thehigh lipid content was found in the ICM region of the blastocyst.Due to the fact that the agreement between SR-IR mappings andFPA-IR imaging show that the use of both HCA and PCA enableus to explore the spectra pattern of inner cell mass, blastocoel

and, outer layer cells. FPA-IR imaging gave very similar results toSR-IR mapping of the blastocyst. Both techniques can be used toapply for many biomedical and biological applications [43,44]where SR-IR mapping can be applied to give results with high spa-tial resolution and high quality information. Such information cancontribute to an understanding of the embryo developmentalmechanism underlying the function of AEA lipid signal moleculemostly found in the ICM region.

Acknowledgements

Authors thank the SLRI for our collaboration, in particular Dr.Mark Tobin, Principle scientist, IR beamline at Australia. Dr. Nich-ada Jearanaikoon, SLRI. Prof. Christopher Wharton, BirminghamUniversity, UK. Special thank to Dr. Peter Lasch.

Fig. 7. AEA signaling in blastocyst activation and implantation. During pre-implantation, High levels of AEA are hydrolyzed by FAAH which is metabolic gatekeeper in cell signaling.

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References

[1] P. Lasch, M. Boese, A. Pacifico, M. Diem, Vibrational Spectroscopy 28 (1) (2002)147–157.

[2] P. Lasch, D. Naumann, Biochimica et Biophysica Acta, Biomembranes 1758 (7)(2006) 814–829.

[3] M. Diem, L. Chiriboga, P. Lasch, A. Pacifico, Biopolymers 67 (4–5) (2002) 349–353.

[4] M. Diem, L. Chiriboga, A. Pacifico, H. Yee, Microbeam Analysis (2000) 77–78.[5] D. Naumann, Infrared spectroscopy in microbiology, in: R.A. Meyers (Ed.),

Encyclopedia of Analytical Chemistry, Copyright Ó John Wiley & Sons Ltd.,2000, pp. 102–131.

[6] D. Naumann, J. Kneipp, C. Kirschner, N.A.N. Thi, P. Lasch, Microbeam Analysis(2000) 61–62.

[7] A. Pacifico, L.A. Chiriboga, P. Lasch, M. Diem, Vibrational Spectroscopy 32 (1)(2003) 107–115.

[8] B. Stuart, D.J. Ando, Biological applications of infrared spectroscopy, Publishedon behalf of ACOL (University of Greenwich) by John Wiley, Chichester, NewYork, 1997.

[9] C. Petibois, B. Drogat, A. Bikfalvi, G. Deleris, M. Moenner, FEBS Letters 581 (28)(2007) 5469–5474.

[10] P. Heraud, S. Caine, G. Sanson, R. Gleadow, B.R. Wood, D. McNaughton, NewPhytologist 173 (1) (2007) 216–225.

[11] F. Bonnier, D. Bertrand, S. Rubin, L. Venteo, M. Pluot, B. Baehrel, M. Manfait,G.D. Sockalingum, Analyst 133 (6) (2008) 784–790.

[12] N.P. Camacho, P. West, P.A. Torzilli, R. Mendelsohn, Biopolymers 62 (1) (2001)1–8.

[13] C. Conti, P. Ferraris, E. Giorgini, C. Rubini, S. Sabbatini, G. Tosi, J.Anastassopoulou, P. Arapantoni, E. Boukaki, S. Konstadoudakis, T.

Theophanides, C. Valavanis, Journal of Molecular Structure 881 (1–3) (2008)46–51.

[14] P. Lasch, W. Haensch, D. Naumann, M. Diem, Biochimica et Biophysica Acta,Molecular Basis of Disease 1688 (2) (2004) 176–186.

[15] P. Dumas, L. Miller, Vibrational Spectroscopy 32 (1) (2003) 3–21.[16] P. Dumas, L. Miller, Journal of Biological Physics 29 (2–3) (2003) 201–218.[17] L.M. Miller, P. Dumas, Biochimica et Biophysica Acta, Biomembranes 1758 (7)

(2006) 846–857.[18] L.M. Miller, P. Dumas, L.M. Miller, N. Jamin, J.L. Teillaud, J.L. Bantignies, G.L.

Carr, Microbeam Analysis Proceedings (2000) 75–76.[19] R.I. Freshney, G.N. Stacey, J.M. Auerback, Interscience, A John Wiley & Sons,

Inc., Publication, 2007.[20] P.H. Lerou, G.Q. Daley, Blood Reviews 19 (6) (2005) 321–331.[21] A. Nagy, K. Vintersten, Methods in Enzymology 418 (2006) 3–21.[22] J. Cai, Y. Zhao, Y.X. Liu, F. Ye, Z.H. Song, H. Qin, S. Meng, Y.Z. Chen, R.D. Zhou, X.J.

Song, Y.S. Guo, M.X. Ding, H. Deng, Hepatology 45 (5) (2007) 1229–1239.[23] T. Cantz, M.P. Manns, M. Ott, Cell and Tissue Research 331 (1) (2008) 271–

282.[24] Z. Wang, H. Lu, Y.C. Wang, X.Q. Cong, Journal of Digestive Diseases 9 (1) (2008)

14–19.[25] M. Maccarrone, M. DeFelici, F.G. Klinger, N. Battista, F. Fezza, E. Dainese, G.

Siracusa, A. Finazzi-Agro, Molecular Human Reproduction 10 (4) (2004) 215–221.

[26] A.H. Taylor, C. Ang, S.C. Bell, J.C. Konje, Human Reproduction Update 13 (5)(2007) 501–513.

[27] H.B. Wang, S.K. Dey, Prostaglandins & Other Lipid Mediators 77 (1–4) (2005)84–102.

[28] H.B. Wang, H.R. Xie, S.K. Dey, AAPS Journal 8 (2) (2006) E425–E432.[29] H.B. Wang, H.R. Xie, X.F. Sun, P.J. Kingsley, L.J. Marnett, B.F. Cravatt, S.K. Dey,

Prostaglandins & Other Lipid Mediators 83 (1–2) (2007) 62–74.[30] H. Schuel, Journal of Clinical Investigation 116 (2006) 2087–2090.[31] Y. Guo, H. Wang, Y. Okamoto, N. Ueda, P.J. Kingsley, L.J. Marnett, H.H.O.

Schmid, S.K. Das, S.K. Dey, Journal of Biological Chemistry 280 (25) (2005)23429–23432.

[32] H.B. Wang, H.R. Xie, S.K. Dey, Journal of Clinical Investigation 16 (2006) 2122–2131.

[33] B.C. Paria, S.K. Das, S.K. Dey, Proceedings of the National academy of Sciencesof the United States of America 92 (1995) 9460–9464.

[34] B.C. Paria, H. Wang, S.K. Dey, Chemistry and Physics of Lipids 121 (1–2) (2002)201–210.

[35] C. Krafft, R. Salzer, S. Seitz, C. Ern, M. Schieker, Analyst 132 (7) (2007) 647–653.[36] D. Ami, T. Neri, A. Natalello, P. Mereghetti, S.M. Doglia, M. Zanoni, M. Zuccotti,

S. Garagna, C.A. Redi, Biochimica et Biophysica Acta, Molecular Cell Research1783 (1) (2008) 98–106.

[37] C.L. Chatot, C.A. Ziomek, B.D. Bavister, J.L. Lewis, I. Torres, Journal ofReproduction and Fertility 86 (2) (1989) 679–688.

[38] P. Lasch, M. Diem, W. Hansch, D. Naumann, Journal of Chemometrics 20 (5)(2006) 209–220.

[39] N.M. Amiali, M.R. Mulvey, B. Berger-Bachi, J. Sedman, A.E. Simor, A.A. Ismail,Journal of Antimicrobial Chemotherapy 61 (1) (2008) 95–102.

[40] W.M. Liu, E.K. Duan, Y.J. Cao, Life Sciences 71 (14) (2002) 1623–1632.[41] M. Maccarrone, Pharmacologyonline 2 (2005) 30–37.[42] M. Maccarrone, British Journal of Pharmacology 153 (2) (2008) 189–198.[43] N.A. Ngo-Thi, C. Kirschner, D. Naumann, Journal of Molecular Structure 661–

662 (2003) 371–380.[44] M.J. Walsh, T.G. Fellous, A. Hammiche, W.R. Lin, N.J. Fullwood, O. Grude, F.

Bahrami, J.M. Nicholson, M. Cotte, J. Susini, H.M. Pollock, M. Brittan, P.L.Martin-Hirsch, M.R. Alison, F.L. Martin, Stem Cells 26 (1) (2008) 108–118.