Paper – Paraffin removal from FFPE sections

Assessment of paraffin removal from prostate FFPE sections using transmission mode FTIR-FPA imaging
Caryn Hughes, Lydia Gaunt, Michael Brown, Noel W. Clarke and Peter Gardner
Anal. Methods (2014)


Formalin-fixed paraffin-embedded (FFPE) tissue sections are routinely analysed for biochemical discrimination by vibrational spectroscopy techniques in the spectral pathology community. In these experiments, it is usually desirable to remove the paraffin from the tissue. This is most commonly performed by the use of xylene but other solvents such as hexane are also used. It is thought that the removal of unbound paraffin wax by such solvents also leaches out lipids native to the tissue, leading to the perception by some that any subsequent analysis on remaining lipids of dewaxed tissue may be unreliable. The scope of the study was to make an assessment of whether a dewaxing protocol can demonstrate that paraffin wax is reliably removed in relation to the detectable limits of transmission mode infrared spectroscopy. Continue reading

Paper – FTIR through glass

Transmission FTIR chemical imaging on glass substrates: applications in infrared spectral histopathology
Paul Bassan, Joe Mellor, Jonathan Shapiro, Kaye J. Williams, Michael Lisanti and Peter Gardner
Anal. Chem. (2014)


Fourier transform infrared chemical imaging in transmission mode has traditionally been performed on expensive mid-IR transparent windows such as barium/calcium fluoride, which are more fragile than glass making preparation in the histopathology laboratories more cumbersome. A solution is presented here by using cheap glass substrates for the FTIR chemical imaging which has a high-wavenumber transmission window allowing measurement of the C–H, N–H and O–H stretches occurring at ca. 2500–3800 cm–1. The “fingerprint” region of the IR spectrum occurring below 1800 cm–1 is not obtainable, however we demonstrate that a wealth of information is contained in the high wavenumber range using 71 patients on a breast tissue microarray (TMA) as a model for investigation. Importantly we demonstrate that the tissue can be classified into four basic tissue cell-types and that using just the epithelial cells, reasonable discrimination of normal and malignant tissue can be found.

Paper – Whole organ, label-free imaging

Whole organ cross-section chemical imaging using label-free mega-mosaic FTIR microscopy
Paul Bassan, Ashwin Sachdeva, Jonathan H. Shanks, Mick D. Brown, Noel W. Clarke and Peter Gardner Analyst (2013)


FTIR chemical imaging has been demonstrated as a promising technique to construct automated systems to complement histopathological evaluation of biomedical tissue samples. The rapid chemical imaging of large areas of tissue has previously been a limiting factor in this application. Consequently, smaller areas of tissue have previously had to be sampled, possibly introducing sampling bias and potentially missing diagnostically important areas. In this report a high spatial resolution chemical image of a whole prostate cross section is shown comprising 66 million pixels. Each pixel represents an area 5.5 × 5.5 μm2 of tissue and contains a full infrared spectrum providing a chemical fingerprint. The data acquisition time was 14 hours, thus showing that a clinical time frame of hours rather than days has been achieved.

Graphical abstract: Whole organ cross-section chemical imaging using label-free mega-mosaic FTIR microscopy

HOT Paper!

Our article on Highlighting a need to distinguish cell cycle signatures from cellular responses to chemotherapeutics in SR-FTIR spectroscopy by Caryn, Frank, Geri and Peter, together with Mick Brown and Noel Clarke from the Paterson Institute for Cancer Research and Kevin Flower from the School of Chemistry, has been awarded Hot Paper status by Analyst.

The article is free to read until 30 November 2012.

Highlighting a need to distinguish cell cycle signatures from cellular responses to chemotherapeutics in SR-FTIR spectroscopy
C. Hughes, M. D. Brown, F. J. Ball, G. Monjardez, N. W. Clarke, K. R. Flower and P. Gardner
Analyst, 2012, 137, 5736-5742
DOI: 10.1039/C2AN35633C


Paper – Rare sub-variants of bladder cancer

FTIR microspectroscopy of selected rare diverse sub-variants of carcinoma of the urinary bladder
Caryn Hughes, Junaid Iqbal-Wahid, Michael Brown, Jonathan H. Shanks, Amanda Eustace, Helen Denley, Peter J. Hoskin, Catharine West, Noel W. Clarke, Peter Gardner
Journal of Biophotonics (2012)


Urothelial carcinomas of the bladder are a heterogeneous group of tumours, although some histological sub-variants are rare and sparsely reported in the literature. Diagnosis of sub-variants from conventional urothelial carcinoma can be challenging, as they may mimic the morphology of other malignancies or benign tumours and therefore their distinction is important. For the first time, the spectral pathology of some of these sub-variants has been documented by infrared microspectroscopy and an attempt made to profile their biochemistry. It is important not only to identify and separate the cancer-associated epithelial tissue spectra from common tissue features such as stroma or blood, but also to detect the signatures of tumour sub-variants. As shown, their spectroscopic signals can change dramatically as a consequence of differentiation. Example cases are discussed and compared with histological evaluations. (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Paper – Problems with transflection-mode IR

The inherent problem of transflection-mode infrared spectroscopic microscopy and the ramifications for biomedical single point and imaging applications
Paul Bassan, Joe Lee, Ashwin Sachdeva, Juliana Pissardini, Konrad M. Dorling, John S. Fletcher, Alex Henderson and Peter Gardner
, 2013, 138, 144-157


Transflection-mode FTIR spectroscopy has become a popular method of measuring spectra from biomedical and other samples due to the relative low cost of substrates compared to transmission windows, and a higher absorbance due to a double pass through the same sample approximately doubling the effective path length. In this publication we state an optical description of samples on multilayer low-e reflective substrates. Using this model we are able to explain in detail the so-called electric-field standing wave effect and rationalise the non-linear change in absorbance with sample thickness. The ramifications of this non-linear change, for imaging and classification systems, where a model is built from tissue sectioned at a particular thickness and compared with tissue of a different thickness are discussed. We show that spectra can be distorted such that classification fails leading to inaccurate tissue segmentation which may have subsequent implications for disease diagnostics applications.

Paper – Cell cycle using a synchrotron

Highlighting a need to distinguish cell cycle signatures from cellular responses to chemotherapeutics in SR-FTIR spectroscopy

C. Hughes, M. D. Brown, F. J. Ball, G. Monjardez, N. W. Clarke, K. R. Flower and P. Gardner

Analyst 137 (2012) 5736-5742


Previous research has seen difficulties in establishing clear discrimination by principal component analysis (PCA) between drug-treated cells analysed by single point SR-FTIR spectroscopy, relative to multisampling cell monolayers by conventional FTIR. It is suggested that the issue arises due to signal mixing between cellular-response signatures and cell cycle phase contributions in individual cells. Consequently, chemometric distinction of cell spectra treated with multiple drugs is difficult even with supervised methods. In an effort to separate cell cycle chemistry from cellular response chemistry in the spectra, renal carcinoma cells were stained with propidium iodide and fluorescent-activated cell sorted (FACS) after exposure to a number of chemotherapeutic compounds; 5-fluorouracil (5FU) and a set of novel gold-based experimental compounds. The cell spectra were analysed separately by PCA in G1, S or G2/M phase. The mode of action of established drug 5FU, known to disrupt S phase, was confirmed by FACS analysis. The chemical signature of 5FU-treated cells discriminated against both the control and gold-compound (KF0101)-treated cell spectra, suggesting a different mode of action due to a difference in cellular response.

Paper – Synchrotron IR for chemotherapeutics in renal cell carcinoma

Investigating cellular responses to novel chemotherapeutics in renal cell carcinoma using SR-FTIR spectroscopy
C. Hughes, M. D. Brown, N. W. Clarke, K. R. Flower and P. Gardner
Analyst 137 (2012) 4720-4726


SR-FTIR spectroscopy was evaluated as a technique to discriminate spectral signals of cellular response at the single cell level, when cancer cells are exposed to chemotherapeutics. 5-Fluorouracil, an established drug of known mode of action, was tested against a renal carcinoma cell line (Caki-2), along with two experimental analogues of gold-based compounds. The use of unsupervised principal component analysis (PCA) failed to clearly define any distinction between control and drug treated cell spectra. Supervised principal component linear discriminant analysis (PC-LDA) did have some potential to reveal signatures of cell response and repair but again failed to distinctly discriminate groups of spectra with different drug treatments. Alternatively, clear PCA discrimination was observed in spectra from average cell populations via single point benchtop spectroscopy, probing several cells simultaneously with an increased aperture. The Caki-2 cell line initially appeared to be sensitive to the novel compounds, inducing a cellular response prior to subsequential cell recovery which was assessed by both PCA and cell viability assays.