Abstract

We describe a new mid-infrared (mid-IR) imaging method specifically designed to augment the H + E tissue staining protocol. Images are taken with bespoke IR filters at wavelengths that enable chemical maps to be generated, corresponding to the cytoplasmic (amide) and nuclear (phosphodiester) components of unstained oesophageal tissue sections. A suitably calibrated combination of these generates false colour computer images that reproduce not only the tissue morphology, but also accurate and quantitative distributions of the nuclear-to-cytoplasmic ratio throughout the tissue section. This parameter is a well documented marker of malignancy, and because the images can be taken and interpreted by clinically trained personnel in a few seconds, we believe this new “digistain” approach makes spectroscopic mid-IR imaging techniques available for the first time as a practical, specific and sensitive augmentation to standard clinical cancer diagnosis methods.

© 2012 OSA

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  1. R. Salzer and H. W. Siesler Eds, (2009) ″Infrared and Raman Spectroscopic imaging″ Wiley-VCH, Weinheim ISBN 978–3-527- 31993–0.
  2. S. J. Jang, J. M. Gardner, and J. Y. Ro, “Diagnostic approach and prognostic factors of cancers,” Adv. Anat. Pathol. 18(2), 165–172 (2011).
    [CrossRef] [PubMed]
  3. L. M. Merlo, L. S. Wang, J. W. Pepper, P. S. Rabinovitch, and C. C. Maley, “Polyploidy, aneuploidy and the evolution of cancer,” Adv. Exp. Med. Biol. 676, 1–13 (2010).
    [CrossRef] [PubMed]
  4. L. Hoover and J. J. Berman, “Epithelial repair versus carcinoma in esophageal brush cytology,” Diagn. Cytopathol. 4(3), 217–223 (1988).
    [CrossRef] [PubMed]
  5. C. N. Battlehner, P. H. Saldiva, C. R. Carvalho, T. Y. Takagaki, G. S. Montes, R. N. Younes, and V. L. Capelozzi, “Nuclear/cytoplasmic ratio correlates strongly with survival in non-disseminated neuroendocrine carcinoma of the lung,” Histopathology 22(1), 31–34 (1993).
    [CrossRef] [PubMed]
  6. D. P. Coco, J. R. Goldblum, J. L. Hornick, G. Y. Lauwers, E. Montgomery, A. Srivastava, H. Wang, and R. D. Odze, “Interobserver variability in the diagnosis of crypt dysplasia in Barrett esophagus,” Am. J. Surg. Pathol. 35(1), 45–54 (2011).
    [CrossRef] [PubMed]

2011 (2)

S. J. Jang, J. M. Gardner, and J. Y. Ro, “Diagnostic approach and prognostic factors of cancers,” Adv. Anat. Pathol. 18(2), 165–172 (2011).
[CrossRef] [PubMed]

D. P. Coco, J. R. Goldblum, J. L. Hornick, G. Y. Lauwers, E. Montgomery, A. Srivastava, H. Wang, and R. D. Odze, “Interobserver variability in the diagnosis of crypt dysplasia in Barrett esophagus,” Am. J. Surg. Pathol. 35(1), 45–54 (2011).
[CrossRef] [PubMed]

2010 (1)

L. M. Merlo, L. S. Wang, J. W. Pepper, P. S. Rabinovitch, and C. C. Maley, “Polyploidy, aneuploidy and the evolution of cancer,” Adv. Exp. Med. Biol. 676, 1–13 (2010).
[CrossRef] [PubMed]

1993 (1)

C. N. Battlehner, P. H. Saldiva, C. R. Carvalho, T. Y. Takagaki, G. S. Montes, R. N. Younes, and V. L. Capelozzi, “Nuclear/cytoplasmic ratio correlates strongly with survival in non-disseminated neuroendocrine carcinoma of the lung,” Histopathology 22(1), 31–34 (1993).
[CrossRef] [PubMed]

1988 (1)

L. Hoover and J. J. Berman, “Epithelial repair versus carcinoma in esophageal brush cytology,” Diagn. Cytopathol. 4(3), 217–223 (1988).
[CrossRef] [PubMed]

Battlehner, C. N.

C. N. Battlehner, P. H. Saldiva, C. R. Carvalho, T. Y. Takagaki, G. S. Montes, R. N. Younes, and V. L. Capelozzi, “Nuclear/cytoplasmic ratio correlates strongly with survival in non-disseminated neuroendocrine carcinoma of the lung,” Histopathology 22(1), 31–34 (1993).
[CrossRef] [PubMed]

Berman, J. J.

L. Hoover and J. J. Berman, “Epithelial repair versus carcinoma in esophageal brush cytology,” Diagn. Cytopathol. 4(3), 217–223 (1988).
[CrossRef] [PubMed]

Capelozzi, V. L.

C. N. Battlehner, P. H. Saldiva, C. R. Carvalho, T. Y. Takagaki, G. S. Montes, R. N. Younes, and V. L. Capelozzi, “Nuclear/cytoplasmic ratio correlates strongly with survival in non-disseminated neuroendocrine carcinoma of the lung,” Histopathology 22(1), 31–34 (1993).
[CrossRef] [PubMed]

Carvalho, C. R.

C. N. Battlehner, P. H. Saldiva, C. R. Carvalho, T. Y. Takagaki, G. S. Montes, R. N. Younes, and V. L. Capelozzi, “Nuclear/cytoplasmic ratio correlates strongly with survival in non-disseminated neuroendocrine carcinoma of the lung,” Histopathology 22(1), 31–34 (1993).
[CrossRef] [PubMed]

Coco, D. P.

D. P. Coco, J. R. Goldblum, J. L. Hornick, G. Y. Lauwers, E. Montgomery, A. Srivastava, H. Wang, and R. D. Odze, “Interobserver variability in the diagnosis of crypt dysplasia in Barrett esophagus,” Am. J. Surg. Pathol. 35(1), 45–54 (2011).
[CrossRef] [PubMed]

Gardner, J. M.

S. J. Jang, J. M. Gardner, and J. Y. Ro, “Diagnostic approach and prognostic factors of cancers,” Adv. Anat. Pathol. 18(2), 165–172 (2011).
[CrossRef] [PubMed]

Goldblum, J. R.

D. P. Coco, J. R. Goldblum, J. L. Hornick, G. Y. Lauwers, E. Montgomery, A. Srivastava, H. Wang, and R. D. Odze, “Interobserver variability in the diagnosis of crypt dysplasia in Barrett esophagus,” Am. J. Surg. Pathol. 35(1), 45–54 (2011).
[CrossRef] [PubMed]

Hoover, L.

L. Hoover and J. J. Berman, “Epithelial repair versus carcinoma in esophageal brush cytology,” Diagn. Cytopathol. 4(3), 217–223 (1988).
[CrossRef] [PubMed]

Hornick, J. L.

D. P. Coco, J. R. Goldblum, J. L. Hornick, G. Y. Lauwers, E. Montgomery, A. Srivastava, H. Wang, and R. D. Odze, “Interobserver variability in the diagnosis of crypt dysplasia in Barrett esophagus,” Am. J. Surg. Pathol. 35(1), 45–54 (2011).
[CrossRef] [PubMed]

Jang, S. J.

S. J. Jang, J. M. Gardner, and J. Y. Ro, “Diagnostic approach and prognostic factors of cancers,” Adv. Anat. Pathol. 18(2), 165–172 (2011).
[CrossRef] [PubMed]

Lauwers, G. Y.

D. P. Coco, J. R. Goldblum, J. L. Hornick, G. Y. Lauwers, E. Montgomery, A. Srivastava, H. Wang, and R. D. Odze, “Interobserver variability in the diagnosis of crypt dysplasia in Barrett esophagus,” Am. J. Surg. Pathol. 35(1), 45–54 (2011).
[CrossRef] [PubMed]

Maley, C. C.

L. M. Merlo, L. S. Wang, J. W. Pepper, P. S. Rabinovitch, and C. C. Maley, “Polyploidy, aneuploidy and the evolution of cancer,” Adv. Exp. Med. Biol. 676, 1–13 (2010).
[CrossRef] [PubMed]

Merlo, L. M.

L. M. Merlo, L. S. Wang, J. W. Pepper, P. S. Rabinovitch, and C. C. Maley, “Polyploidy, aneuploidy and the evolution of cancer,” Adv. Exp. Med. Biol. 676, 1–13 (2010).
[CrossRef] [PubMed]

Montes, G. S.

C. N. Battlehner, P. H. Saldiva, C. R. Carvalho, T. Y. Takagaki, G. S. Montes, R. N. Younes, and V. L. Capelozzi, “Nuclear/cytoplasmic ratio correlates strongly with survival in non-disseminated neuroendocrine carcinoma of the lung,” Histopathology 22(1), 31–34 (1993).
[CrossRef] [PubMed]

Montgomery, E.

D. P. Coco, J. R. Goldblum, J. L. Hornick, G. Y. Lauwers, E. Montgomery, A. Srivastava, H. Wang, and R. D. Odze, “Interobserver variability in the diagnosis of crypt dysplasia in Barrett esophagus,” Am. J. Surg. Pathol. 35(1), 45–54 (2011).
[CrossRef] [PubMed]

Odze, R. D.

D. P. Coco, J. R. Goldblum, J. L. Hornick, G. Y. Lauwers, E. Montgomery, A. Srivastava, H. Wang, and R. D. Odze, “Interobserver variability in the diagnosis of crypt dysplasia in Barrett esophagus,” Am. J. Surg. Pathol. 35(1), 45–54 (2011).
[CrossRef] [PubMed]

Pepper, J. W.

L. M. Merlo, L. S. Wang, J. W. Pepper, P. S. Rabinovitch, and C. C. Maley, “Polyploidy, aneuploidy and the evolution of cancer,” Adv. Exp. Med. Biol. 676, 1–13 (2010).
[CrossRef] [PubMed]

Rabinovitch, P. S.

L. M. Merlo, L. S. Wang, J. W. Pepper, P. S. Rabinovitch, and C. C. Maley, “Polyploidy, aneuploidy and the evolution of cancer,” Adv. Exp. Med. Biol. 676, 1–13 (2010).
[CrossRef] [PubMed]

Ro, J. Y.

S. J. Jang, J. M. Gardner, and J. Y. Ro, “Diagnostic approach and prognostic factors of cancers,” Adv. Anat. Pathol. 18(2), 165–172 (2011).
[CrossRef] [PubMed]

Saldiva, P. H.

C. N. Battlehner, P. H. Saldiva, C. R. Carvalho, T. Y. Takagaki, G. S. Montes, R. N. Younes, and V. L. Capelozzi, “Nuclear/cytoplasmic ratio correlates strongly with survival in non-disseminated neuroendocrine carcinoma of the lung,” Histopathology 22(1), 31–34 (1993).
[CrossRef] [PubMed]

Srivastava, A.

D. P. Coco, J. R. Goldblum, J. L. Hornick, G. Y. Lauwers, E. Montgomery, A. Srivastava, H. Wang, and R. D. Odze, “Interobserver variability in the diagnosis of crypt dysplasia in Barrett esophagus,” Am. J. Surg. Pathol. 35(1), 45–54 (2011).
[CrossRef] [PubMed]

Takagaki, T. Y.

C. N. Battlehner, P. H. Saldiva, C. R. Carvalho, T. Y. Takagaki, G. S. Montes, R. N. Younes, and V. L. Capelozzi, “Nuclear/cytoplasmic ratio correlates strongly with survival in non-disseminated neuroendocrine carcinoma of the lung,” Histopathology 22(1), 31–34 (1993).
[CrossRef] [PubMed]

Wang, H.

D. P. Coco, J. R. Goldblum, J. L. Hornick, G. Y. Lauwers, E. Montgomery, A. Srivastava, H. Wang, and R. D. Odze, “Interobserver variability in the diagnosis of crypt dysplasia in Barrett esophagus,” Am. J. Surg. Pathol. 35(1), 45–54 (2011).
[CrossRef] [PubMed]

Wang, L. S.

L. M. Merlo, L. S. Wang, J. W. Pepper, P. S. Rabinovitch, and C. C. Maley, “Polyploidy, aneuploidy and the evolution of cancer,” Adv. Exp. Med. Biol. 676, 1–13 (2010).
[CrossRef] [PubMed]

Younes, R. N.

C. N. Battlehner, P. H. Saldiva, C. R. Carvalho, T. Y. Takagaki, G. S. Montes, R. N. Younes, and V. L. Capelozzi, “Nuclear/cytoplasmic ratio correlates strongly with survival in non-disseminated neuroendocrine carcinoma of the lung,” Histopathology 22(1), 31–34 (1993).
[CrossRef] [PubMed]

Adv. Anat. Pathol. (1)

S. J. Jang, J. M. Gardner, and J. Y. Ro, “Diagnostic approach and prognostic factors of cancers,” Adv. Anat. Pathol. 18(2), 165–172 (2011).
[CrossRef] [PubMed]

Adv. Exp. Med. Biol. (1)

L. M. Merlo, L. S. Wang, J. W. Pepper, P. S. Rabinovitch, and C. C. Maley, “Polyploidy, aneuploidy and the evolution of cancer,” Adv. Exp. Med. Biol. 676, 1–13 (2010).
[CrossRef] [PubMed]

Am. J. Surg. Pathol. (1)

D. P. Coco, J. R. Goldblum, J. L. Hornick, G. Y. Lauwers, E. Montgomery, A. Srivastava, H. Wang, and R. D. Odze, “Interobserver variability in the diagnosis of crypt dysplasia in Barrett esophagus,” Am. J. Surg. Pathol. 35(1), 45–54 (2011).
[CrossRef] [PubMed]

Diagn. Cytopathol. (1)

L. Hoover and J. J. Berman, “Epithelial repair versus carcinoma in esophageal brush cytology,” Diagn. Cytopathol. 4(3), 217–223 (1988).
[CrossRef] [PubMed]

Histopathology (1)

C. N. Battlehner, P. H. Saldiva, C. R. Carvalho, T. Y. Takagaki, G. S. Montes, R. N. Younes, and V. L. Capelozzi, “Nuclear/cytoplasmic ratio correlates strongly with survival in non-disseminated neuroendocrine carcinoma of the lung,” Histopathology 22(1), 31–34 (1993).
[CrossRef] [PubMed]

Other (1)

R. Salzer and H. W. Siesler Eds, (2009) ″Infrared and Raman Spectroscopic imaging″ Wiley-VCH, Weinheim ISBN 978–3-527- 31993–0.

Cited By

OSA participates in CrossRef's Cited-By Linking service. Citing articles from OSA journals and other participating publishers are listed here.

Alert me when this article is cited.


Figures (6)

Fig. 1
Fig. 1

Schematics of the optical setup. A Barium Fluoride condenser (f = 37.5mm) lens was placed between the Globar element and sample as shown to provide a sample illumination as uniform as possible. The f~50mm imaging lens was placed on a translational stage to allow focussing adjustments (needed to accommodate wavelength dispersion in the imaging lens) alongside the electronically operated shutter and narrow bandpass filters. The IR camera was placed in the image plane of the lens and interfaced with Labview to record data

Fig. 2
Fig. 2

(Lower trace) Area-averaged absorbance spectrum of a 2.5mm x 2.5mm section of an unstained oesophageal biopsy section (sample 5601) recorded using a Jasco FTIR microscope at a spectral resolution of 4cm−1. (Upper trace), measured transmission spectra of the 4 bespoke IR bandpass filters that were used in this study to obtain signal and baseline images at wavelengths corresponding to absorption by the Amide I and Phosphodiester chemical moieties. Variations in the areas of the transmission peaks were carefully measured and the respective Spectroscopic Image (SI) data arrays were scaled accordingly.

Fig. 3
Fig. 3

A USAF 1951 Test Resolution Pattern imaged at a wavelength of λ~6.00µm with the use of the appropriate narrow bandpass filter. By determining the smallest line pair elements resolvable on screen (Group 5; Set 2- 36.0 line pairs/mm) the useful spatial resolution of the imaging system was estimated at r~14µm.

Fig. 4
Fig. 4

(a) Visible light image of a adjacent section taken from sample 5601, stained with a standard H+E protocol, (b) False colour chemical map from an adjacent, unstained section from the same biopsy sample, plotted to show the distribution of the concentration of the Amide moiety. This image is acquired by subtracting the image acquired through the λ = 6.23µm bandpass filter from that acquired at λ = 6.00µm.

Fig. 5
Fig. 5

Plot of the mean absorbance of selected rectangular areas of tissue, with widely varying nuclear density in samples 5601 and 5933 versus the areal density of nuclei found in that area in the corresponding H & E stained section. These areas are shown highlighted in Fig. 6. The strong linear correlation argues that the main contribution to the latter comes dominantly from PO2- moieties in the phosphodiester groups in DNA and less from free ADP/ATP. The vertical errors are smaller than the symbol sizes in all cases.

Fig. 6
Fig. 6

(a) and (c): false-colour chemical maps plotted to represent the ratio of the concentrations of the amide and phosphodiester moieties in the unstained sections of biopsy samples 5601 and 5933 respectively. (b) and (d):- standard H+E stained sections from adjacent sections of the same two biopsies, for comparison. Note the pronounced increase in the relative DNA concentration in the high grade dysplasia area (delineated by the dotted line) of the sample 5933 compared with the healthy epithelial tissue (delineated by the solid line). Tissue regions were delineated by eye.

Tables (1)

Tables Icon

Table 1 Characteristics of the Bespoke IR Bandpass Filters Used

Equations (6)

Equations on this page are rendered with MathJax. Learn more.

r = 0.61 λ N A
R i j O i j = X i j
B i j O i j = Y i j
T i j = X i j Y i j
A i j = log 10 T i j
A P i j = C i j N i j = A i j ( 6.00 μ m ) A i j ( 6.23 μ m ) A i j ( 8.13 μ m ) A i j ( 8.50 μ m )

Metrics