Lipid droplets imaging with three-photon microscopy

Lipid droplets(LDs)participate in many physiological processes,the abnormality of which will cause chronic diseases and pathologies such as diabetes and obesity.It is crucial to monitor the distribution of LDs at high spatial resolution and large depth.Herein,we carried three-photon imaging of LDs in fat liver.Owing to the large three-photon absorption cross-section of the luminogen named NAP-CF_(3)(1:67×10^(-79) cm^(6) s^(2)),three-photon fluorescence fat liver imaging reached the largest depth of 80μm.Fat liver diagnosis was successfully carried out with excellent performance,providing great potential for LDs-associated pathologies research.

Constructing D-π-A-π dye to obtain red-emission fluorescent probe for structured illumination microscopy imaging of lipid droplet dynamics

Lipid droplets(LDs)are dynamic organelles interacting with a variety of intracellular organelles.Tracking intracellular LD dynamics employing synthetic small molecules is crucial for biological studies.Fluorescence imaging in the red and near infrared(NIR)region is more suitable for biological imaging due to its low phototoxicity and high signal-to-noise ratio.However,available LD-dyes in the red region with remarkable environmental sensitivity,selectivity for LDs staining are limited.Here,we constructed a red-emission D-π-A-π type LDdye LD 688P with higher environmental sensitivity and suitable“calculated log P”(Clog P)for LDs dynamic imaging.LD 688P was proved to be highly selective and photostable for tracing LD fusion including multiple consecutive fusions and fusions in a centrosymmetric manner by super-resolution microscopy.We believe that the D-π-A-π skeleton would be an efficient strategy to construct red and even NIR-emission dyes.

Mild synthesis of ultra-bright carbon dots with solvatochromism for rapid lipid droplet monitoring in varied physiological processes

Lipid droplets(LDs)participating in various cellular activities and are increasingly being emphasized.Fluorescence imaging provides powerful tool for dynamic tracking of LDs,however,most current LDs probes remain inconsistent performance such as low Photoluminescence Quantum Yield(PLQY),poor photostability and tedious washing procedures.Herein,a novel yellow-emissive carbon dot(OT-cD)has been synthesized conveniently with high PLQY up to 90%.Besides,OT-CD exhibits remarkable amphiphilicity and solvatochromic property with lipid-water partition coefficient higher than 2,which is much higher than most LDs probes.These characters enable OT-CD high brightness,stable and wash-free LDs probing,and feasible for in vivo imaging.Then,detailed observation of LDs morphological and polarity variation dynamically in different cellular states were recorded,including ferroptosis and other diseases processes.Furthermore,fast whole imaging of zebrafish and identifed LD enrichment in injured liver indicate its further feasibility for in vivo application.In contrast to the reported studies to date,this approach provides a versatile conventional synthesis system for high-performance LDs targeting probes,combing the advantages of easy and high-yield production,as well as robust brightness and stability for long-term imaging,facilitating investigations into organelle interactions and LD-associated diseases.

Facile synthesis of ultrabright luminogens with specific lipid droplets targeting feature for in vivo two-photon fluorescence retina imaging

The application of fluorescent probes for in vivo retinal imaging is of great importance,which could provide direct and crucial imaging evidence for a better understanding of common eye diseases.Herein,a group of bright organic luminogens with typical electron-donating(D)and electron-accepting(A)structures(abbreviated as LDs-BDM,LDs-BTM,and LDs-BHM)was synthesized through a simple single-step reaction.They were found to be efficient solid-state emitters with high fluorescence quantum yields of above 70%(e.g.,83.7%for LDs-BTM).Their light-emission properties could be tuned by the modulation ofπ-conjugation effect with methoxy groups at different substituent positions.Their resulting fluorescent nanoparticles(NPs)were demonstrated as specific lipid droplets(LDs)targeting probes with high brightness,good biocompatibility,and satisfactory photostability.LDs-BTM NPs with a large two-photon absorption cross section(σ2=249 GM)were further utilized as ultrabright two-photon fluorescence(2PF)nanoprobes for in vivo retina imaging of live zebrafish by NIR excitation at an ultralow concentration(0.5μmol/L).Integrated histological structures at the tissue level and corresponding fine details at the cellular level of the embryonic retina of live zebrafish were clearly demonstrated.This is the first report of using ultrabright LDs-targeting nanoprobes to accurately measure fine details in the retina with 2PF microscopic technique.These good results are anticipated to open up a new avenue in the development of efficient 2PF emitters for non-invasive bioimaging of living animals.

脂滴荧光探针的研究进展

脂滴是由中性脂核心和镶嵌有表面蛋白的磷脂单分子层组成的高度动态细胞器,在细胞内发挥着调节能量平衡和脂质代谢等重要生理作用。研究表明脂滴的功能障碍与多种疾病直接相关,包括非酒精性脂肪肝、动脉粥样硬化和癌症等,这就迫切需要能精准识别脂滴的工具以监测其变化过程。荧光探针因其快速响应和实时检测能力而成为脂滴动态成像的理想工具。本文总结了检测与其他细胞器相互作用和相关疾病的脂滴探针,比较了不同设计策略的优缺点,也讨论了当前脂滴探针面临的一些挑战与发展趋势,为未来脂滴探针的开发提供了理论依据。

BODIPY 493 acts as a bright buffering fluorogenic probe for super-resolution imaging of lipid droplet dynamics

The need for temporal resolution and long-term stability in super-resolution fluorescence imaging has motivated research to improve the photostability of fluorescent probes.Due to the inevitable photobleaching of fluorophores,it is difficult to obtain long-term super-resolution imaging regardless of the self-healing strategy of introducing peroxide scavengers or the strategy of fluorophore structure modification to suppress TICT formation.The buffered fluorogenic probe uses the intact probes in the buffer pool to continuously replace the photobleached ones in the target,which greatly improves the photostability and enables stable dynamic super-resolution imaging for a long time.But the buffering capacity comes at the expense of reducing the number of fluorescent probes in targets,resulting in low staining fluorescence intensity.In this paper,we selected BODIPY 493,a lipid droplet probe with high fluorescence brightness,to explore the dynamic process of lipid droplet staining of this probe in cells.We found that BODIPY 493 only needs very low laser power for lipid droplet imaging due to the high molecular accumulation in lipid droplets and the high brightness,and the spatiotemporal resolution is greatly improved.More importantly,we found that BODIPY 493 also has a certain buffering capacity,which enables BODIPY 493 to be used for super-resolution imaging of lipid droplet dynamics.This work reminds researchers to coordinate the buffering capacity and brightness of fluorogenic probes.

脂滴荧光标记原理及其特性的比较分析

脂滴(Lipid droplets,LDs)是细胞内储存中性脂质的细胞器,参与脂质代谢、膜合成转运、信号转导和蛋白质降解等多种重要生理过程。处于动态循环的LDs常与多种细胞器共同行使功能,因此高效的LDs成像技术成为生物学研究及医学病理诊断的有力工具。本文阐述了LDs荧光标记原理,并针对多种荧光染料的特点进行了详细介绍与比较分析,旨在为LDs荧光染料的特异性选用提供参考。

用于细胞脂滴超分辨荧光成像的有机荧光探针研究进展

脂滴是真核细胞中必不可少的一种球形细胞器,与很多细胞生理学过程息息相关。荧光成像技术是观察研究脂滴最有力的工具之一。受光学衍射极限的限制,传统的宽场以及共聚焦显微镜所能达到的成像分辨率约为250 nm左右,这对于观测小脂滴,尤其是新生脂滴(尺寸约30~60 nm)来说是远远不够的。在这种情况下,近年来新兴的各种能够打破衍射极限的超分辨荧光显微镜(如受激发射损耗显微镜、结构光照明显微镜以及光激活定位显微镜等)逐渐吸引了科研人员的兴趣。为了得到高分辨率脂滴荧光图像,除了上述超分辨显微镜之外,还需要具有与之相匹配的高性能荧光探针。本文将简要介绍这几种超分辨显微镜的工作原理,讨论其对荧光探针光物理性质的特殊要求,并进一步系统总结脂滴超分辨成像荧光探针的研究进展。与此同时,本文将分析对比不同超分辨显微镜在脂滴荧光成像方面的优势与不足,并对其发展趋势进行展望。

脂质响应型探针用于动脉粥样硬化成像及治疗的研究进展

动脉粥样硬化是心血管疾病的主要威胁之一,可能导致如急性心肌梗死在内的急性冠状动脉综合征、卒中,甚至猝死等一系列严重的不良后果.脂质的异常积累是动脉粥样硬化的标志之一,有必要观察它们在体内的数量、定位和分布.荧光探针具有可操作性强、高时空分辨率和与活体生物相容性佳的优点,有望成为了解动脉粥样硬化中的脂质功能的有利工具.本文综合评述了基于罗丹明、香豆素、氟化硼二吡咯(BODIPY)、1,8-萘酰亚胺等有机框架结构以及金属配合物的脂滴特异性光学成像探针的研究进展,并重点介绍了其在动脉粥样硬化斑块成像、手术导航及治疗等领域的应用.最后,对该研究领域进行了总结和展望,希望可为相关研究提供有益参考.

Red-emissive carbon quantum dots minimize phototoxicity for rapid and long-term lipid droplet monitoring

Long-term fluorescence monitoring of subcellular organelles is crucial for cellular physiology and pathology studies.Lipid droplets(LDs)are increasingly recognized for their involvement in various biological processes,to influence disease development through diverse behaviors However,existing LD probes face challenges in achieving high targeting and long-term monitoring due to poor photostability and long-term phototoxicity.Carbon quantum dots(CQDs)have gained prominence due to their exceptional fluorescence properties,but their prevalent blue excitation wavelength presents difficulties for long-term imaging.Herein,we synthesized red-emissive carbon quantum dot(R-CQDs)with superior photobleaching resistance and red-emission,thus enabling harmlessly fluorescence monitoring of cells longer than3 h.In addition,R-CQD exhibits suitable amphiphilicity and remarkable solvatochromic effect,allowing rapid targeting to LDs for immediate imaging without cumbersome washing steps.Hence,R-CQD shows high performance for extended observation of dynamic LD behavior in various biological processes,which is confirmed by documenting the course of LDs during starvation as well as lipotoxicity.Compared to commercial probes,R-CQD extends live cell imaging time by at least 9-fold,facilitating the study of LD behavioral characteristics under diverse physiological or pathological conditions.This work provides a reliable fluorescence tool for tracking intercellular microenvironment dynamically thus to understand the divers biological or disease mechanism.

具有荧光开关特性的喹啉衍生物用于高效选择性标记细胞脂滴

脂滴是一种重要的细胞器,与细胞中多种生理活动密切相关。为了观察脂滴并研究其多种多样的功能,共聚焦荧光成像技术是最有力的工具之一。然而,细胞脂滴荧光成像所需的具有高荧光亮度和高标记选择性的脂滴荧光探针却十分有限,这严重限制了脂滴的深入研究。研制了一种具有荧光开关特性的喹啉衍生物脂滴荧光探针(Lipi-QL)。该探针因其敏感的极性淬灭荧光特性,展现出了很高的脂滴标记选择性。Lipi-QL能靶向脂滴是因为其脂溶性,进入脂滴后,荧光增强是脂滴内的较低极性引起的。给受体型分子结构还赋予了该探针高的荧光亮度以及大的斯托克斯位移。将该探针用于细胞脂滴共聚焦荧光成像,在不同浓度下都实现了显著优于脂滴商用探针BODIPY 493/503的标记选择性。此外,使用该荧光探针实现了固定细胞的三维共聚焦成像和活细胞的四色共聚焦成像。该荧光探针的研制一方面为脂滴生理功能的研究提供了强有力的工具,另一方面也为新型高标记选择性荧光探针的设计提供了新的思路。

一种快速分析细胞内脂质沉积的方法

目的:建立快速分析细胞内脂质沉积的方法。方法:以油酸刺激人肝癌细胞形成脂质沉积,利用BODIPY 493/503染色细胞内脂滴,在Matlab环境下以荧光图像分析技术分析脂滴荧光强度;采用蛋白印迹法分析脂质沉积标志蛋白——脂滴表面蛋白Adipophilin的蛋白水平,以及采用实时荧光定量PCR法检测Adipophilin的基因表达水平,以验证荧光图像分析技术的可靠性。结果:利用荧光图像分析技术建立了快速定量脂质沉积的方法,结果显示该方法下400μmol/L油酸显著提高细胞内脂质含量达1.41倍,与标志蛋白Adipophilin的蛋白水平增高2.07倍,基因表达水平增高1.53倍一致。结论:该可快速定量细胞内脂质沉积水平,且结果可靠。