Palmer Gregory M, Marshek Crystal L, Vrotsos Kristin M, Ramanujam Nirmala
Department of Biomedical Engineering, University of Wisconsin, Madison, Wisconsin 53706, USA.
Lasers Surg Med. 2002;30(3):191-200. doi: 10.1002/lsm.10026.
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.
STUDY DESIGN/MATERIALS AND 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.
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).
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.
在开发用于体内检测癌前病变和癌症的荧光光谱系统时，通常需要对组织活检样本进行初步测试。当前的标准方案要求组织在活检后立即冷冻，随后解冻以进行光谱分析，但这一过程可能会对组织的光谱特性产生深远影响。本研究旨在探究用于体外荧光光谱研究的最佳组织处理方法。
研究设计/材料与方法：本研究采用金黄叙利亚仓鼠颊囊的上皮组织。进行了三项具体实验。首先，在跨越紫外 - 可见（UV - VIS）光谱的多个激发波长下，对体内组织以及冷冻和解冻后的组织活检样本的荧光特性进行了表征。其次，在对应于先前确定的荧光峰的激发波长下，系统地比较了体内、离体（活检后立即）以及冷冻和解冻后的组织荧光发射光谱。最后，测量了对应于荧光峰的激发和发射波长对处的强度随活检后时间的变化。还进行了UV - VIS光谱范围内的漫反射测量，以评估在这些不同激发 - 发射波长下，氧合、血容量和散射对组织荧光的影响。
本研究表明，在300至700nm的整个UV - VIS范围内，相对于体内荧光发射光谱数据，冷冻和解冻过程在强度和线形上产生了显著偏差。相比之下，对离体发射光谱的检查表明，除了在500至700nm之间外，它紧密保留了体内发射光谱的强度和线形。观察到的偏差可以通过漫反射测量来解释，这表明离体组织中血红蛋白去氧增加以及散射随波长的变化，并且冷冻和解冻样本中总血红蛋白吸收增加。此外，发现在1.5小时的时间窗口内，由于活检或其他因素导致的组织降解所引起的光谱变化（强度变化10 - 30%）明显小于与冷冻和解冻过程相关的变化（强度降低50 - 70%）。
研究发现，冷冻和解冻对组织荧光特性的影响大于活检后90分钟内组织降解所带来的任何变化。在合理的时间窗口内进行离体荧光测量，对于金黄仓鼠颊囊组织而言，具有更准确地再现临床相关体内情况的优势。因此，在组织活检研究中，组织样本在测量前理想情况下应保持未冷冻状态。
