Sakhalkar H S, Dewhirst M, Oliver T, Cao Y, Oldham M
Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710, USA.
Phys Med Biol. 2007 Apr 21;52(8):2035-54. doi: 10.1088/0031-9155/52/8/001. Epub 2007 Mar 20.
Optical emission computed tomography (optical-ECT) is a technique for imaging the three-dimensional (3D) distribution of fluorescent probes in biological tissue specimens with high contrast and spatial resolution. In optical-ECT, functional information can be imaged by (i) systemic application of functional labels (e.g. fluorophore labelled proteins) and/or (ii) endogenous expression of fluorescent reporter proteins (e.g. red fluorescent protein (RFP), green fluorescent protein (GFP)) in vivo. An essential prerequisite for optical-ECT is optical clearing, a procedure where tissue specimens are made transparent to light by sequential perfusion with fixing, dehydrating and clearing agents. In this study, we investigate clearing protocols involving a selection of common fixing (4% buffered paraformaldehyde (PFA), methanol and ethanol), dehydrating (methanol and ethanol) and clearing agents (methyl salicylate and benzyl-alcohol-benzyl-benzoate (BABB)) in order to determine a 'fluorescence friendly' clearing procedure. Cell culture experiments were employed to optimize the sequence of chemical treatments that best preserve fluorescence. Texas red (TxRed), fluorescein isothiocyanate (FITC), RFP and GFP were tested as fluorophores and fluorescent reporter proteins of interest. Fluorescent and control cells were imaged on a microscope using a DSred2 and FITC filter set. The most promising clearing protocols of cell culture experiments were applied to whole xenograft tumour specimens, to test their effectiveness in large unsectioned samples. Fluorescence of TxRed/FITC fluorophores was not found to be significantly affected by any of the test clearing protocols. RFP and GFP fluorescence, however, was found to be significantly greater when cell fixation was in ethanol. Fixation in either PFA or methanol resulted in diminished fluorescence. After ethanol fixation, the RFP and GFP fluorescence proved remarkably robust to subsequent exposure to either methyl salicylate or BABB. The optimized optical clearing procedure of ethanol fixation followed by methyl salicylate clearing preserved the fluorescence of constitutive RFP in whole xenograft tumour specimens, about 1 cc in dimension, indicating successful extension from cell plating experiments to whole tissue samples. Finally, the feasibility of imaging the 3D distribution of viable tumour cells (as indicated by the RFP emission) is demonstrated by optical-ECT imaging of cleared xenograft tumours using an in-house system.
光学发射计算机断层扫描（optical-ECT）是一种用于对生物组织样本中荧光探针的三维（3D）分布进行成像的技术，具有高对比度和空间分辨率。在光学发射计算机断层扫描中，功能信息可以通过以下方式成像：（i）全身应用功能标记物（例如荧光团标记的蛋白质）和/或（ii）荧光报告蛋白（例如红色荧光蛋白（RFP）、绿色荧光蛋白（GFP））在体内的内源性表达。光学发射计算机断层扫描的一个基本前提是光学透明化，这是一个通过依次用固定剂、脱水剂和透明剂灌注使组织样本对光透明的过程。在本研究中，我们研究了涉及选择常见固定剂（4%缓冲多聚甲醛（PFA）、甲醇和乙醇）、脱水剂（甲醇和乙醇）和透明剂（水杨酸甲酯和苄醇 - 苯甲酸苄酯（BABB））的透明化方案，以确定一种“荧光友好”的透明化程序。采用细胞培养实验来优化能最佳保留荧光的化学处理顺序。测试了德克萨斯红（TxRed）、异硫氰酸荧光素（FITC）、RFP和GFP作为感兴趣的荧光团和荧光报告蛋白。使用DSred2和FITC滤光片组在显微镜下对荧光细胞和对照细胞进行成像。将细胞培养实验中最有前景的透明化方案应用于整个异种移植肿瘤标本，以测试它们在未切片的大样本中的有效性。未发现任何测试的透明化方案对TxRed/FITC荧光团的荧光有显著影响。然而，当细胞固定在乙醇中时，发现RFP和GFP荧光明显更强。用PFA或甲醇固定会导致荧光减弱。乙醇固定后，RFP和GFP荧光对随后暴露于水杨酸甲酯或BABB具有显著的抗性。乙醇固定后用水杨酸甲酯透明化的优化光学透明化程序保留了整个尺寸约为1立方厘米的异种移植肿瘤标本中组成型RFP的荧光，表明从细胞铺板实验成功扩展到了整个组织样本。最后，使用内部系统对清除后的异种移植肿瘤进行光学发射计算机断层扫描成像，证明了对存活肿瘤细胞的3D分布（由RFP发射指示）进行成像的可行性。
