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

摘要 癌症是一种极其复杂和动态的疾病， 生物学已经发展，人们意识到需要更多的分子信息来 表征异质性肿瘤内的多种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理解， 诊断，可以揭示潜在的驱动机制，可以针对病人护理。 这种需求推动了当前大规模“组学”技术的趋势，例如next。 代测序和质谱，但这些方法不提供细胞分辨率或 了解异质系统和识别罕见细胞类型所需的直接功能细节。 基于Sers和质谱细胞术的成像平台具有实现极高的成像质量的潜力。 许多独特的探头，但具有实际问题，即采集时间长和固有的 技术复杂性将限制未来的临床应用。荧光成像是最广泛的 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 使用具有不同光谱特性的探针，但 需要在复用容量方面的显著改进。荧光寿命是一种特性， 扩大荧光成像的多路复用能力，但迄今为止，这种方法仅限于 最多两个物种，因为缺乏兼容的探针。在这里，我们寻求发展荧光 终身成像显微镜（FLIM）成为高内涵的肿瘤分子分析平台 同时保持传统光谱荧光成像的速度和简单性。到 为了实现这一目标，我们将创建第一个荧光“寿命探针库”，它将在 相同的光谱窗口，但表现出独特的荧光寿命衰减， 强大的相量方法我们将通过创建一个新的探测器类来填充生存期库 它们以模块化、灵活的纳米颗粒形式容纳不同的组件。具体来说，我们将 将不同的荧光成分以精确控制的比例封装在二氧化硅纳米颗粒内， 或外壳，这将使我们能够调整探针寿命，而不影响发射光谱。这种硅基 该方法将使合成和生物缀合程序标准化，通过 高负载能力、生物相容性和保护细胞免受潜在毒性荧光组分的影响。 重要的是，二氧化硅壳还将保护荧光组分免受环境影响， 锁定信号属性。我们将首先使用四种不同荧光物质的面板，其具有相似的荧光强度。 黄色的发射光谱，但独特的固有寿命，并建立定量的方法 解析癌细胞系的分子表达水平。接下来，我们将创建可调纳米探针 并构建一个具有最佳兼容寿命的库，我们预计至少包括7个 纳米探针最后，我们将把我们的可调纳米探针框架扩展到4个额外的光谱窗口， 从而形成具有35个检测通道的组合寿命和光谱成像平台， 一项使用人类前列腺肿瘤标本的初步研究。我们的基于荧光寿命的分子 成像平台将是高度多路复用的，同时还保持速度和简单性， 传统的荧光成像，这将有助于推动转化到临床竞技场。我们的平台 还将与活的或固定的标本、诊断组织切片、甚至体内 成像应用。这种功能、速度、简单性和灵活性的结合目前还不是 可用于其他高含量分子分析平台。

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.