Novel Fluorophores for Molecular and Cellular Imaging

用于分子和细胞成像的新型荧光团

• 批准号: 8604153
• 负责人: ZYGMUNT GRYCZYNSKI
• 金额: $ 43.57万
• 依托单位: UNIVERSITY OF NORTH TEXAS HLTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2017-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8604153
• 关键词: 2-cyclopentyl-5-(5-isoquinolylsulfonyl)-6-nitro-1H-benzo(D)imidazole; Anisotropy; Binding; Biological Process; Cancer Detection; Cell Culture Techniques; Cells; Cellular Membrane; Chemistry; Communication; Complex; DNA; Detection; Development; Disease; Drops; Dyes; Energy Transfer; Event; Extinction (Psychology); Fluorescence; Fluorescence Anisotropy; Fluorescence Polarization; Goals; Image; Interest Group; Kinetics; Label; Lanthanoid Series Elements; Lasers; Life; Living Donors; Malignant Neoplasms; Matrix Metalloproteinases; Measurement; Measures; Medical; Methodology; Methods; Molecular; Monitor; Organ; Pathway interactions; Peptides; Photons; Physiologic pulse; Physiological; Process; Property; Proteins; RNA; Reporting; Resolution; Sampling; Sensitivity and Specificity; Signal Transduction; Source; System; Technology; Testing; Time; Tissues; Universities; absorption; base; bioimaging; biological systems; cellular imaging; chemical property; drug testing; fluorophore; imaging modality; improved; in vivo; molecular dynamics; molecular imaging; molecular mass; molecular/cellular imaging; nanoscience; nanosecond; novel; outcome forecast; protein protein interaction; public health relevance; quantum; response; single molecule; time use; two-photon; whole body imaging

DESCRIPTION (provided by applicant): At the core of biological function lays the ability of proteins to interact and associate with each other and the imaging methods capable of reporting on molecular events and interactions with high sensitivity and high resolution become indispensible. Fluorescence methods have a potential for highly sensitive detection and become essential for studying molecular processes with high specificity and sensitivity through a variety of signaling mechanisms. Tens of fluorescence probes are developed every year to be used for proteins/DNA/RNA labeling and to study molecular pathways and interactions as well as tissue imaging. However after many years of significant effort we are still missing "perfect" probes. There are many obstacles for markers that can be used for studying biological processes in cells and tissue. Two fundamental common problems for biomedical imaging, from single molecule studies and cellular imaging to whole body imaging are the background signal and availability of highly bright probes with suitable fluorescence lifetimes. The background signal (sample autofluorescence, scattering, and non- specific probe binding) always compromise sensitivity and specificity. The need for imaging kinetics and dynamics of molecular interactions/processes (like protein-protein interactions) requires probes with fluorescence lifetimes comparable to the mobility of interacting molecular partners. Many membrane and cellular proteins are large with molecular masses from 20 kDa to much over 100 kDa for which tumbling time and conformational changes are within tens and hundreds of nanoseconds. Within the large arsenal of dyes available today we have many bright fluorophores with fluorescence lifetimes of few nanoseconds or less and some luminophores like lanthanides with exited state lifetimes in microseconds. At present we lack fluorophores in red spectral range with fluorescence lifetimes over 10 ns. In this application we propose to utilize and further develop new group of small organic compounds [1,2]. The new group of azaoxa-triangulenium dyes offers excellent physico-chemical properties that will have unprecedented impact on molecular imaging. The rigid and small triangular frame of this organic compound has very favorable spectral properties including high photostability and most importantly unprecedented long single exponential fluorescence lifetime (~20 ns). We now propose to develop active and enhanced forms of these compounds to be used for studying molecular processes and interactions on a single molecule level, cellular level, and tissue imaging. In parallel to dyes development and tuning their spectral properties we will develop novel methodologies based on time gated detection to eliminate background signal and study dynamics of molecular processes and interactions by fluorescence polarization and FRET. This will enable: (1) precise time-resolved imaging that brings dynamic information about observed processes in large molecular complexes not available from steady-state measurements; (2) use of time-gated detection that will dramatically decrease background and improve imaging sensitivity over 100 folds; (3) new molecular beacon-type probes based on FRET, long lived donor, and time-gated detection that will have enormous signal gain of 105. In addition, 10-30 ns fluorescence lifetimes are much longer from the lifetime of typical background signal and in the same time easy for gating. Importantly time-resolved measurements for such lifetimes can be comfortably made with a pulsed laser source with a repetition rate of 1-5 MHz, in contrast it will require significantly longer time to collect enough photons in case of the lanthanides where the repetition rates are only in kHz.

描述（由申请人提供）：在生物学功能的核心中，蛋白质彼此相互作用和相关的能力以及能够报告分子事件和具有高灵敏度和高分辨率的分子事件和相互作用的成像方法变得不可或缺。荧光方法具有高度敏感检测的潜力，对于通过各种信号机制研究具有高特异性和敏感性的分子过程至关重要。每年都会开发数十个荧光探针，用于蛋白质/DNA/RNA标记，并研究分子途径和相互作用以及组织成像。但是，经过多年的巨大努力，我们仍然缺少“完美”的探针。标记有许多障碍，可用于研究细胞和组织中的生物学过程。从单分子研究和细胞成像到全身成像的两个基本常见问题是背景信号和具有合适荧光寿命的高度明亮探针的可用性。背景信号（样品自动荧光，散射和非特异性探针结合）始终会损害灵敏度和特异性。对分子相互作用/过程的动力学成像动力学的需求（例如蛋白质 - 蛋白质相互作用）需要探针，其荧光寿命与相互作用的分子伴侣的迁移率相当。许多膜和细胞蛋白较大，分子质量从20 kDa到超过100 kDa，而翻滚时间和构象变化在数十万和数百纳米秒之内。在当今可用的大型染料库中，我们有许多明亮的荧光团，荧光寿命很少或更少或更少，以及一些发光液（如兰塔尼德），如兰植物，在微秒中占据了状态寿命。目前，我们缺乏红色光谱范围内的荧光团，荧光寿命超过10 ns。 在此应用中，我们建议利用并进一步开发新的小有机化合物[1,2]。新的Azaoxa-Triangulenium染料组提供了出色的物理化学特性，这些特性将对分子成像产生前所未有的影响。该有机化合物的刚性和小三角形框架具有非常有利的光谱特性，包括高光稳定性和最重要的是前所未有的长单指数荧光寿命（〜20 ns）。现在，我们建议开发这些化合物的主动和增强形式，用于在单分子水平，细胞水平和组织成像上研究分子过程和相互作用。 在染料开发和调整光谱特性的同时，我们将根据时间门控检测开发新的方法，以消除背景信号和研究分子过程的动力学以及通过荧光极化和FRET进行的分子过程和相互作用的动力学。这将启用：（1）精确的时间分辨成像，这些成像带来了有关在稳态测量中无法获得的大分子复合物中观察到的过程的动态信息； （2）使用时间门控检测，该检测将大大降低背景并提高100倍的成像灵敏度； （3）基于FRET，长期生存的供体和时间门控的检测的新分子信标探针，其巨大信号增益为105。此外，从典型的背景信号的寿命中，10-30 NS荧光寿命比典型的荧光寿命更长同时容易进行门控。重要的是，这种一生的时间分辨测量值可以通过重复率为1-5 MHz的脉冲激光源舒适地制成，相比之下，在仅在重复速率的情况下，需要更长的时间来收集足够的光子在KHz。

项目成果

Polarization properties of fluorescent BSA protected Au25 nanoclusters.

• DOI: 10.1039/c3nr34152f
• 发表时间: 2013-04-21
• 期刊: Nanoscale
• 影响因子: 6.7
• 作者: Raut S;Chib R;Rich R;Shumilov D;Gryczynski Z;Gryczynski I
• 通讯作者: Gryczynski I

Linear dependence of the water proton transverse relaxation rate on the shear modulus of hydrogels.

水质子横向弛豫率对水凝胶剪切模量的线性依赖性。

• DOI: 10.1039/c4cc04717f
• 发表时间: 2014-10-18
• 期刊: Chemical communications (Cambridge, England)
• 作者: Feng Y;Taraban MB;Yu YB
• 通讯作者: Yu YB

BSA Au clusters as a probe for enhanced fluorescence detection using multipulse excitation scheme.

BSA Au 簇作为探针，使用多脉冲激发方案增强荧光检测。

• DOI: 10.2174/1389201015666140523161038
• 发表时间: 2014
• 期刊: Current pharmaceutical biotechnology
• 影响因子: 2.8
• 作者: Raut,SangramL;Rich,Ryan;Fudala,Rafal;Kokate,R;Kimball,JD;Borejdo,Julian;Vishwanatha,JamboorK;Gryczynski,Zygmunt;Gryczynski,Ignacy
• 通讯作者: Gryczynski,Ignacy

Fluorescent Polyelectrolyte Capped Silver Nanoclusters: Optimization and Spectroscopic Evaluation.

• DOI: 10.1016/j.cplett.2012.08.042
• 发表时间: 2012-10-11
• 期刊: CHEMICAL PHYSICS LETTERS
• 影响因子: 2.8
• 作者: Raut, Sangram;Rich, Ryan;Akopova, Irina;Thyrhaug, Erling;Shtoyko, Tanya;Shumilov, Dimytro;Gryczynski, Zygmunt;Gryczynski, Ignacy
• 通讯作者: Gryczynski, Ignacy

Enhanced DNA detection using a multiple pulse pumping scheme with time-gating (MPPTG).

使用具有时间选通 (MPPTG) 的多脉冲泵浦方案增强 DNA 检测。

• DOI: 10.1039/c8an00136g
• 发表时间: 2018
• 期刊: The Analyst
• 作者: Kimball,JosephD;Maliwal,Badri;Raut,SangramL;Doan,Hung;Nurekeyev,Zhangatay;Gryczynski,Ignacy;Gryczynski,Zygmunt
• 通讯作者: Gryczynski,Zygmunt