Transforming fluorescence lifetime imaging microscopy into a fast and simple platform for high-content molecular analysis

将荧光寿命成像显微镜转变为快速、简单的高内涵分子分析平台

• 批准号: 9320961
• 负责人: Jered Brackston Haun
• 金额: $ 26.08万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9320961
• 关键词: Adoption; Affect; Androgen Receptor; Biological; Biological Process; Cancer Biology; Cancer Diagnostics; Cancer cell line; Cells; Clinical; Color; Complex; Cytometry; Detection; Diagnostic; Disease; Encapsulated; Energy Transfer; Engineering; Exhibits; Fluorescence; Foundations; Future; Goals; Human; Indolent; Knowledge; Libraries; Light; Malignant Neoplasms; Malignant neoplasm of prostate; Mass Spectrum Analysis; Methodology; Methods; Molecular; Molecular Analysis; Molecular Medicine; Molecular Probes; Molecular Target; Optics; Patient Care; Phase; Pilot Projects; Procedures; Property; Prostatic Neoplasms; Resolution; Sampling; Signal Transduction; Silicon Dioxide; Specimen; Speed; System; Techniques; Time; Tissues; Translating; Translations; Tumor Biology; Tumor Markers; amorphous silicate; base; biomaterial compatibility; cancer type; cell type; flexibility; fluorescence imaging; fluorescence lifetime imaging; imaging platform; improved; in vivo imaging; innovation; molecular diagnostics; molecular imaging; nanoparticle; nanoprobe; neoplastic cell; next generation sequencing; novel; optical spectra; prospective; public health relevance; spectrograph; trend; tumor

ABSTRACT Cancer is an exceedingly complex and dynamic disease, and as our knowledge of tumor biology has grown, so has the realization that ever more molecular information is needed to characterize the diverse array of functional states and cell types within heterogeneous tumors. Extracting this information would aid our basic understanding of cancer biology and enable molecular diagnostics that could reveal the underlying driver mechanisms that could be targeted for patient care. This need has driven the current trend towards massive scale “omics” techniques, such as next generation sequencing and mass spectrometry, but these methods do not offer the cellular resolution or direct functional detail necessary to understand heterogenous systems and identify rare cell types. Imaging platforms based on SERS and mass cytometry have the potential to achieve extremely high numbers of unique probes, but have practical issues in the form of long acquisition times and inherent technological complexity that will limit future clinical adoption. Fluorescence imaging is the most widely used detection technique in biological and clinical settings, and enables fast and simple detection of upwards of ten molecular targets using probes that have different spectral properties. However, a drastic improvement in multiplexing capacity is needed. Fluorescence lifetime is a property that could expand the multiplexing capacity of fluorescence imaging, but to date this approach has been limited to at most two species due to the lack of compatible probes. Here we seek to develop fluorescence lifetime imaging microscopy (FLIM) into a high-content molecular analysis platform from tumor specimens while maintaining the speed and simplicity of traditional spectral fluorescence imaging. To achieve this goal, we will create the first fluorescence “lifetime probe libraries,” which will emit light in the same spectral window but exhibit unique fluorescence lifetime decays that can be resolved using the powerful phasor approach. We will populate our lifetime libraries by creating a new class of probes that house different components in a modular, flexible nanoparticle format. Specifically, we will encapsulate different fluorescent components at precisely controlled ratios within a silica nanoparticle or shell, which will allow us to tune probe lifetime without affecting emission spectra. This silica-based approach will normalize synthesis and bioconjugation procedures, maximize signal intensity through high loading capacity, be biocompatible, and shield cells from potentially toxic fluorescent components. Critically, the silica shell will also protect the fluorescent components from environmental effects, locking in signal properties. We will first use a panel of four different fluorescent species with similar yellow emission spectra but unique intrinsic lifetimes, and establish methodologies for quantitatively resolving molecular expression levels of cancer cell lines. Next we will create our tunable nanoprobes and construct a library with optimally compatible lifetimes, which we expect will include at least 7 nanoprobes. Finally, we will extend our tunable nanoprobe framework to 4 additional spectral windows, resulting in a combined lifetime and spectral imaging platform with 35 detection channels, and perform a pilot study using human prostate tumor specimens. Our fluorescence lifetime-based molecular imaging platform will be both highly multiplexed while also maintaining the speed and simplicity of traditional fluorescence imaging, which should help drive translation into the clinical arena. Our platform will also be compatible with live or fixed specimens, diagnostic tissue sections, and even in vivo imaging applications. This combination of power, speed, simplicity, and flexibility is not currently available in other high-content molecular analysis platforms.

抽象的 癌症是一种极其复杂且动态的疾病，随着我们对肿瘤的了解 生物学已经发展，人们也意识到需要更多的分子信息来 表征异质性肿瘤内不同的功能状态和细胞类型。 提取这些信息将有助于我们对癌症生物学的基本了解，并使分子生物学成为可能 可以揭示潜在驱动机制的诊断，可以作为患者护理的目标。 这种需求推动了当前大规模“组学”技术的趋势，例如下一步 世代测序和质谱分析，但这些方法不提供细胞分辨率或 了解异质系统和识别稀有细胞类型所需的直接功能细节。 基于SERS和质谱流式技术的成像平台有潜力实现极高的 大量独特的探针，但存在采集时间长和固有的实际问题 技术复杂性将限制未来的临床应用。 在生物和临床环境中使用检测技术，并能够快速、简单地检测 然而，使用具有不同光谱特性的探针来检测十个以上的分子目标。 荧光寿命是一个可以实现的特性，需要大幅提高。 扩大荧光成像的多重能力，但迄今为止这种方法仅限于 由于缺乏兼容的探针，最多只能有两个物种。在这里，我们寻求开发荧光。 将终生成像显微镜 (FLIM) 转变为肿瘤的高内涵分子分析平台 样本，同时保持传统光谱荧光成像的速度和简单性。 为了实现这一目标，我们将创建第一个荧光“终身探针库”，它将在 相同的光谱窗口，但表现出独特的荧光寿命衰减，可以使用 我们将通过创建一类新的探针来填充我们的生命周期库。 具体来说，我们将采用模块化、灵活的纳米颗粒形式容纳不同的组件。 以精确控制的比例将不同的荧光成分封装在二氧化硅纳米颗粒内 或外壳，这将使我们能够在不影响发射光谱的情况下调整探针寿命。 该方法将使合成和生物共轭程序标准化，通过最大化信号强度 高负载能力，具有生物相容性，并保护细胞免受潜在有毒荧光成分的影响。 至关重要的是，二氧化硅壳还将保护荧光成分免受环境影响， 我们将首先使用具有相似特征的四种不同荧光物质的组合。 黄色发射光谱但独特的内在寿命，并建立定量方法 接下来我们将创建可调谐纳米探针。 并构建一个具有最佳兼容生命周期的库，我们预计该库将至少包含 7 个 最后，我们将可调谐纳米探针框架扩展到 4 个额外的光谱窗口， 形成具有 35 个检测通道的组合寿命和光谱成像平台，并执行 使用人类前列腺肿瘤样本进行的一项试点研究。 成像平台将高度复用，同时保持速度和简单性 传统的荧光成像，这应该有助于推动我们的平台转化为临床领域。 也将与活体或固定标本、诊断组织切片甚至体内兼容 这种功能、速度、简单性和灵活性的结合目前尚不具备。 可用于其他高内涵分子分析平台。