Padera Timothy P, Stoll Brian R, So Peter T, Jain Rakesh K
Massachusetts General Hospital, Boston, MA 02114, USA.
Mol Imaging. 2002 Jan-Mar;1(1):9-15. doi: 10.1162/15353500200200004.
The ability to determine various functions of genes in an intact host will be an important advance in the postgenomic era. Intravital imaging of gene regulation and the physiological effect of the gene products can play a powerful role in this pursuit. Intravital epifluorescence microscopy has provided powerful insight into gene expression, tissue pH, tissue pO2, angiogenesis, blood vessel permeability, leukocyte-endothelial (L-E) interaction, molecular diffusion, convection and binding, and barriers to the delivery of molecular and cellular medicine. Multiphoton laser scanning microscopy (MPLSM) has recently been applied in vivo to overcome three drawbacks associated with traditional epifluorescence microscopy: (i) limited depth of imaging due to scattering of excitation and emission light; (ii) projection of three-dimensional structures onto a two-dimensional plane; and (iii) phototoxicity. Here, we use MPLSM for the first time to obtain high-resolution images of deep tissue lymphatic vessels and show an increased accuracy in quantifying lymphatic size. We also demonstrate the use of MPLSM to perform accurate calculations of the volume density of angiogenic vessels and discuss how this technique may be used to assess the potential of antiangiogenic treatments. Finally, high-speed MPLSM, applied for the first time in vivo, is used to compare L-E interactions in normal tissue and a rhabdomyosarcoma tumor. Our work demonstrates the potential of MPLSM to noninvasively monitor physiology and pathophysiology both at the tissue and cellular level. Future applications will include the use of MPLSM in combination with fluorescent reporters to give novel insight into the regulation and function of genes.
在完整宿主中确定基因各种功能的能力将是后基因组时代的一项重要进展。对基因调控和基因产物生理效应进行活体成像在这一探索过程中可发挥重要作用。活体落射荧光显微镜已为深入了解基因表达、组织pH值、组织氧分压、血管生成、血管通透性、白细胞 - 内皮细胞（L - E）相互作用、分子扩散、对流与结合以及分子和细胞药物递送障碍提供了有力帮助。多光子激光扫描显微镜（MPLSM）最近已应用于活体，以克服与传统落射荧光显微镜相关的三个缺点：（i）由于激发光和发射光的散射导致成像深度有限；（ii）三维结构投影到二维平面上；（iii）光毒性。在此，我们首次使用MPLSM获得深部组织淋巴管的高分辨率图像，并显示在量化淋巴管大小时准确性有所提高。我们还展示了使用MPLSM对血管生成血管的体积密度进行精确计算，并讨论了该技术如何用于评估抗血管生成治疗的潜力。最后，首次在活体中应用的高速MPLSM用于比较正常组织和横纹肌肉瘤肿瘤中的L - E相互作用。我们的工作证明了MPLSM在组织和细胞水平上无创监测生理和病理生理的潜力。未来的应用将包括将MPLSM与荧光报告基因结合使用，以深入了解基因的调控和功能。
