Cell Biochemistry and Biophysics

, Volume 53, Issue 3, pp 145–157 | Cite as

Fluorescence Lifetime Imaging of Endogenous Fluorophores in Histopathology Sections Reveals Differences Between Normal and Tumor Epithelium in Carcinoma In Situ of the Breast

  • Matthew W. Conklin
  • Paolo P. Provenzano
  • Kevin W. Eliceiri
  • Ruth Sullivan
  • Patricia J. Keely
Original Paper


The classical examination of histology slides from a mouse model of breast cancer has been extended in this study to incorporate modern multiphoton excitation and photon-counting techniques. The advantage of such approaches is quantification of potential diagnostic parameters from the fluorescence emission signal, whereby the traditional descriptive staging process is complemented by measurements of fluorescence intensity, lifetime, and spectra. We explored whether the clinical “gold standard” of eosin and hematoxylin stained histology slides would provide optical biomarker signatures of diagnostic value. Alternatively, we examined unstained slides for changes in intensity and/or fluorescence lifetime of relevant endogenous fluorophores. Although eosin provided a strong emission signal and had distinct spectra and lifetime, we found that it was not useful as a fluorescent biological marker, particularly when combined with hematoxylin. Instead, we found that the properties of the fluorescence from the endogenous fluorophores NADH and FAD were indicative of the pathological state of the tissue. Comparing regions of carcinoma in situ to adjacent histologically normal regions, we found that tumor cells produced higher intensity and had a longer fluorescence lifetime. By imaging at 780 nm and 890 nm excitation, we were able to differentiate the fluorescence of FAD from NADH by separating the emission spectra. The shift to a longer lifetime in tumor cells was independent of the free or bound state of FAD and NADH, and of the excitation wavelength. Most forms of cancer have altered metabolism and redox ratios; here we present a method that has potential for early detection of these changes, which are preserved in fixed tissue samples such as classic histopathology slides.


Fluorescence lifetime FAD NADH Multiphoton microscopy Carcinoma in situ Breast cancer 

Supplementary material

12013_2009_9046_MOESM1_ESM.pdf (36 kb)
Supplemental Figure 1 Endogenous fluorescence was preserved in histopathology slides. MCF10A cells cultured in a 3 mg/mL collagen gel were imaged in live samples (upper panels) that were then fixed, paraffin-embedded, and mounted to slides (lower panels). MCF10A cells imaged in fixed, sectioned samples (lower panels) maintained the endogenous fluorophores, and lifetime values, of those noted in live MCF10A cells (upper panels). The acini imaged in gels is not only similar in appearance to groups of cells in slides cut from the same gel, they have similar fluorescence lifetime (seen in the table below, n = number of total images analyzed from live and fixed tissue from 6 experiments) and identical spectra. An excitation wavelength of 780 nm was used and data was mapped using the same lifetime color range, scale bar is 50 μm. (PDF 37 kb)


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Copyright information

© Humana Press Inc. 2009

Authors and Affiliations

  • Matthew W. Conklin
    • 1
    • 2
    • 3
  • Paolo P. Provenzano
    • 1
    • 2
    • 3
  • Kevin W. Eliceiri
    • 2
  • Ruth Sullivan
    • 2
    • 3
  • Patricia J. Keely
    • 1
    • 2
    • 3
  1. 1.Department of PharmacologyUniversity of WisconsinMadisonUSA
  2. 2.Laboratory for Optical and Computational Instrumentation (LOCI), Laboratory for Molecular BiologyUniversity of WisconsinMadisonUSA
  3. 3.Paul P. Carbone Comprehensive Cancer CenterUniversity of WisconsinMadisonUSA

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