Near-IR Bioluminescence Imaging

描述（由申请人提供）：萤火虫荧光素酶的生物发光成像（BLI）作为一种强大、廉价和非侵入性的方法，在整个生物体的背景下监测基因表达、酶活性、蛋白质-蛋白质相互作用和蛋白质降解，已被广泛接受。相对于PET和MRI等其他成像方式，生物发光成像具有成本低、速度快、灵敏度高、通量高、非专业人员易于使用等优点。这些优点使BLI成为快速评估肿瘤进展和对潜在治疗反应的首选方法。BLI的主要限制是可见光穿透组织的能力差。被照射的组织对近红外光（650-900 nm）是最透明的，其中自身荧光、散射和血红蛋白对可见光的吸收是最小的。尽管花费了大量的努力来分离和诱变发光蛋白，但没有一种荧光素酶能最大地发出50 - 650 nm的光。我们提出了两种协同方法来将萤火虫荧光素酶的光输出转移到近红外：1)共振能量转移到可靶向的近红外荧光团，以及2)合成新型荧光素底物，最大限度地发出更长波长的光。这些目标与体内肿瘤成像相关，因为它们将提高生物发光成像的速度、检测极限和深度穿透。将表达发光蛋白的癌细胞引入小鼠体内，使研究人员能够监测活体动物的癌症进展和对药物治疗的反应。然而，发射的光被血液强烈吸收，因此不会在老鼠体内传播很远。我们正在改变发射光的波长，以避免被血液吸收，并允许光线更深地穿透小鼠，从而提高我们检测小肿瘤或深部器官肿瘤的能力。

DESCRIPTION (provided by applicant): Bioluminescent imaging (BLI) with firefly luciferase has gained widespread acceptance as a powerful, inexpensive, and non-invasive method to monitor gene expression, enzymatic activity, protein-protein interactions and protein degradation in the context of the whole organism. Relative to other imaging modalities such as PET and MRI, bioluminescence imaging has the advantages of low cost, speed, sensitivity, high throughput and ease of use by non-specialists. These advantages make BLI the method of choice for rapidly assessing tumor progression and response to potential therapeutics. The major limitation of BLI is the poor penetration of visible light through tissue. Illuminated tissue is most transparent to near-IR light (650-900 nm), where autofluorescence, scattering, and the absorption of visible light by hemoglobin is minimal. Despite considerable effort spent isolating and mutagenizing luminescent proteins, there is no luciferase that maximally emits light >650 nm. We propose two synergistic approaches to shift the light output of firefly luciferase to the near-IR: 1) Resonance energy transfer to a targetable near-IR fluorophore, and 2) Synthesis of novel luciferin substrates that maximally emit light at longer wavelengths. These aims are relevant to in vivo cancer imaging because they will improve the speed, detection limit, and depth penetration of bioluminescence imaging. Introduction of cancer cells that express light-emitting proteins into mice has allowed researchers to monitor cancer progression and response to drug treatment in living animals. However, the emitted light is strongly absorbed by blood, and thus does not travel far through the mouse. We are changing the wavelength of the emitted light to avoid absorption by blood and allow deeper penetration of the light through the mouse, improving our ability to detect small tumors or tumors in deep organs.