Multispectral imaging of clinically relevant cellular targets in tonsil and lymphoid tissue using semiconductor quantum dots
Multispectral imaging of clinically relevant cellular targets in tonsil and lymphoid tissue using semiconductor quantum dots
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DOI：
10.1038/modpathol.3800628
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发表时间：
2006-09-01
期刊：
影响因子：
7.5
通讯作者：
Pittaluga, Stefania
中科院分区：
文献类型：
--
作者：
Fountaine, Thomas J.;Wincovitch, Stephen M.;Pittaluga, Stefania
对患者组织标本中分子表位或抗原的表达和空间分布的测定，大大提高了病理学家对疾病过程进行分类的能力。某些疾病的病理生理学特征是发育控制抗原的表达增加或减少，这些抗原被定义为分化标记物簇，目前形成了理解淋巴系统恶性肿瘤的基础。虽然发色原和有机荧光团已经在免疫组织化学分析中使用了一段时间，但合成的无机荧光团半导体，即量子点，提供了一种通用的替代报告系统。量子点是稳定的荧光团，耐光漂白，具有宽的激发范围和窄的发射光谱。到目前为止，常规处理、福尔马林固定的组织只能同时用两个量子点记者进行探测。在本研究中，具有不同发射光谱的链霉亲和素标记的量子点被用于识别各种差异表达的抗原(表面、细胞质和核)。使用共聚焦激光扫描显微镜对载玻片进行分析，这使得使用单一激发波长(488 nm Ar激光)能够检测到多达7个信号(链霉亲和素共轭量子点525、565、585、605、655、705和805 nm)，以及使用调谐到760 nm的红外激光进行双光子激发的4‘6-二氨基-2-苯基吲哚的检测。这些信号中的每一个都是特定于预期的形态免疫组织化学靶点的。此外，被测试的七个链霉亲和素标记的量子点中的五个(不是链霉亲和素标记的量子点585或805 nm)用于同一组织切片，并可在常规处理的福尔马林固定的石蜡包埋切片上同时进行分析。这种多路传输方法的应用将使研究人员能够同时探索疾病背后与临床相关的多维细胞相互作用。
Determination of the expression and spatial distribution of molecular epitopes, or antigens, in patient tissue specimens has substantially improved the pathologist's ability to classify disease processes. Certain disease pathophysiologies are marked by characteristic increased or decreased expression of developmentally controlled antigens, defined as Cluster of Differentiation markers, that currently form the foundation for understanding lymphoid malignancies. While chromogens and organic fluorophores have been utilitized for some time in immunohistochemical analyses, developments in synthetic, inorganic fluorophore semiconductors, namely quantum dots, offer a versatile alternative reporter system. Quantum dots are stable fluorophores, are resistant to photobleaching, and are attributed with wide excitation ranges and narrow emission spectra. To date, routinely processed, formalin-fixed tissues have only been probed with two quantum dot reporters simultaneously. In the present study, streptavidin-conjugated quantum dots with distinct emission spectra were tested for their utility in identifying a variety of differentially expressed antigens (surface, cytoplasmic, and nuclear). Slides were analyzed using confocal laser scanning microscopy, which enabled with a single excitation wavelength (488 nm argon laser) the detection of up to seven signals (streptavidin-conjugated quantum dots 525, 565, 585, 605, 655, 705 and 805 nm) plus the detection of 4'6-DiAmidino-2-Phenyllndole with an infra-red laser tuned to 760 nm for two photon excitation. Each of these signals was specific for the intended morphologic immunohistochemical target. In addition, five of the seven streptavidin-conjugated quantum dots tested (not streptavidin-conjugated quantum dots 585 or 805 nm) were used on the same tissue section and could be analyzed simultaneously on routinely processed formalin-fixed, paraffin-embedded sections. Application of this multiplexing method will enable investigators to explore the clinically relevant multidimensional cellular interactions that underlie diseases, simultaneously.