title: Optimal methods for fluorescence and diffuse reflectance measurements of tissue biopsy samples.
abstract: OBJECTIVE
  In developing fluorescence spectroscopy systems for the in vivo detection of pre-cancer and cancer, it is often necessary to perform preliminary testing on tissue biopsies. Current standard protocols call for the tissue to be immediately frozen after biopsy and later thawed for spectroscopic analysis, but this process can have profound effects on the spectroscopic properties of tissue. This study investigates the optimal tissue handling methods for in vitro fluorescence spectroscopy studies.
  
  
  METHODS
  The epithelial tissue of the Golden Syrian hamster cheek pouch was used in this study. Three specific experiments were carried out. First, the fluorescence properties of tissues in vivo and of frozen and thawed tissue biopsies were characterized at multiple excitation wavelengths spanning the ultraviolet-visible (UV-VIS) spectrum. Next, comparison of tissue fluorescence emission spectra in vivo, ex vivo (immediately after biopsy), and after the freeze and thaw process were systematically carried out at the excitation wavelengths corresponding to the previously identified fluorescence peaks. Lastly, intensities at the excitation and emission wavelength pairs corresponding to the fluorescence peaks were measured as a function of time after biopsy. Diffuse reflectance measurements over the UV-VIS spectrum were also made to evaluate the effects of oxygenation, blood volume, and scattering on the tissue fluorescence at these different excitation-emission wavelengths.
  
  
  RESULTS
  This study indicates that the freezing and thawing process produces a significant deviation in intensity and lineshape relative to the in vivo fluorescence emission spectral data over the entire UV-VIS range between 300 and 700 nm. By contrast, examination of ex vivo emission spectra reveals that it closely preserves both the intensity and lineshape of the in vivo emission spectra except between 500 and 700 nm. The observed deviations can be explained by the diffuse reflectance measurements, which suggest increased hemoglobin deoxygenation and wavelength dependent changes in scattering in ex vivo tissues, and increased total hemoglobin absorption in the frozen and thawed samples. Furthermore, it was found that over a time window of 1.5 hours, spectroscopic changes brought about by degradation of the tissue due to biopsy or other factors are significantly smaller (10-30% variations in intensity) than those associated with the freezing and thawing process (50-70% decrease in intensity).
  
  
  CONCLUSIONS
  It was found that the effects of freezing and thawing on the fluorescence properties of tissue are greater than any changes brought about by degradation of tissue over a time frame of 90 minutes after biopsy. Performing ex vivo fluorescence measurements within a reasonable time window has the advantage of more accurately reproducing the clinically relevant in vivo conditions in the case of the hamster cheek pouch tissue. Therefore, in tissue biopsy studies, the tissue sample should ideally be maintained in an unfrozen state prior to measurement.
authors.affiliation: Department of Biomedical Engineering, University of Wisconsin, Madison, Wisconsin 53706, USA.
authors.forename: Gregory M
authors.initials: GM
authors.lastname: Palmer
authors.forename: Crystal L
authors.initials: CL
authors.lastname: Marshek
authors.forename: Kristin M
authors.initials: KM
authors.lastname: Vrotsos
authors.forename: Nirmala
authors.initials: N
authors.lastname: Ramanujam
mesh_terms: D000818:Animals; D001706:Biopsy; D002610:Cheek; D006224:Cricetinae; D008647:Mesocricetus; D009061:Mouth Mucosa / Q000166:cytology*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013048:Specimen Handling; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods*