Development of a dual-wavelength fluorescent nanoprobe for in vivo and in vitro cell tracking consecutively

Many imaging probes have been developed for a wide variety of imaging modalities. However, no optical imaging probe could be utilized for both microscopic and whole animal imaging. To fill the gap, the dual-wavelength fluorescent imaging nanoprobe was developed to simultaneously carry both visible-range fluorescent dye and near-infrared (NIR) dye. Emission scan confirms that the nanoprobe exhibits two separate peaks with strong fluorescent intensity in both visible and NIR ranges. Furthermore, the dual-wavelength fluorescent nanoprobe has high photostability and colloidal stability, as well as long shelf-life. In vitro cell culture experiments show that the nanoprobe has the ability to label different types of cells (namely, esophageal, prostate, fibroblast and macrophage cell) for fluorescent microscope imaging. More importantly, cell tracking experiments confirm that cell migration and distribution in various organs can be tracked in real time using in vivo whole-body NIR imaging and in vitro microscopic imaging, respectively.     

Fluorescent probes for bioimaging applications

Fluorescent probes based on small organic molecules have become indispensable tools in modern biology because they provide dynamic information concerning the localization and quantity of the molecules of interest, without the need of genetic engineering of the sample. In this review, following a brief outline of the principle of fluorescence imaging, we recount some recent achievements in the field of small-molecular fluorescent probes. First, probes for metal cations, including those suitable for two-photon imaging, are introduced. Next, methodologies to visualize proteases are discussed, with special emphasis on activity-based probes for use in vivo. All these probes have been confirmed to be applicable to cellular or in vivo imaging.     

Going live: A comparative analysis of the suitability of the RFP derivatives RedStar,mCherry and tdTomato for intravital and in vitro live imaging of Plasmodium parasites

Fluorescent proteins have proven to be important tools for in vitro live imaging of parasites and for imaging of parasites within the living host by intravital microscopy. We observed that a red fluorescent transgenic malaria parasite of rodents, Plasmodium berghei-RedStar, is suitable for in vitro live imaging experiments but bleaches rapidly upon illumination in intravital imaging experiments using mice. We have therefore generated two additional transgenic parasite lines expressing the novel red fluorescent proteins tdTomato and mCherry, which have been reported to be much more photostable than first- and second-generation red fluorescent proteins including RedStar. We have compared all three red fluorescent parasite lines for their use in in vitro live and intravital imaging of P. berghei blood and liver parasite stages, using both confocal and wide-field microscopy. While tdTomato bleached almost as rapidly as RedStar, mCherry showed improved photostability and was bright in all experiments performed.     

含Dsred类似生色团的红色荧光蛋白的发展及应用

自从绿色荧光蛋白(GFP)被发现以来,荧光蛋白在生物医学领域已经成为一种重要的荧光成像工具．随着红色荧光蛋白DsRed的出现,各种优化的DsRed突变体和远红荧光蛋白也不断涌现．其中荧光蛋白生色团的形成机制对改建更优的荧光蛋白变种影响很大,对于红色荧光蛋白而言,大多数的红色荧光蛋白的生色团类型为DsRed类似生色团,在此基础上又出现了Far-red DsRed类似生色团．目前,含DsRed类似生色团的荧光蛋白主要有单体红色荧光蛋白、光转换荧光蛋白、斯托克斯红移蛋白、荧光计时器等．这些优化的荧光蛋白作为分子探针可以实现对活细胞、细胞器或胞内分子的时空标记和追踪,已经在生物工程学、细胞生物学、基础医学领域得到广泛应用．本文综述了含DsRed类似生色团的荧光蛋白的研究进展及其应用,以及由此发展起来的远红荧光蛋白在活体显微成像技术中的应用,并展望了荧光探针技术研究的新方向．     

Functional in vivo imaging of cysteine cathepsin activity in murine model of inflammation

Near-infrared fluorophore (NIRF)-labeled imaging probes are becoming increasingly important in bio-molecular imaging applications, that is, in animal models for tumor imaging or inflammation studies. In this study we showed that the previously introduced chemical concept of ‘Reverse Design’ represents an efficient strategy for the generation of selective probes for cysteine proteases from chemically optimized protease inhibitors for investigations in proteomic lysates as well as for in vivo molecular imaging studies. The newly developed activity-based probe AW-091 was demonstrated to be highly selective for cathepsin S in vitro and proved useful in monitoring cysteine cathepsin activity in vivo, that is, in zymosan-induced mouse model of inflammation. AW-091 showed higher signal-to-background ratios at earlier time points than the commercially available polymer-based ProSense680 (VisEn Medical) and thus represents an efficient new tool for studying early proteolytic processes leading to various diseases, including inflammation, cancer, and rheumatoid arthritis. In addition, the fluorescent signal originating from the cleaved AW-091 was shown to be reduced by the administration of an anti-inflammatory drug, dexamethasone and by the cathepsin inhibitor E-64, providing a valuable system for the evaluation of small-molecule inhibitors of cathepsins.     

Quantification and its Applications in Fluorescent Microscopy Imaging

Fluorescent microscope imaging technologies have developed at a rapid pace in recent years. High-throughput 2D fluorescent imaging platforms are now in wide use and are being applied on a proteome wide scale. Multiple fluorophore 3D imaging of live cells is being used to give detailed localization and subcellular structure information. Further, 2D and 3D video microscopy are giving important insights into the dynamics of protein localization and transport. In parallel with these developments, significant research has gone into developing new methodologies for quantifying and extracting meaning from the imaging data. Here we outline and give entry points to the literature on approaches to quantification such as segmentation, tracking, automated classification and data visualization. Particular attention is paid to the distinction between and application of concrete quantification measures such as number of objects in a cell, and abstract measures such as texture.     

荧光蛋白研究进展

荧光蛋白在生物学众多研究领域中有着广泛的应用,基于荧光蛋白的分子探针和标记方法已成为活细胞或活体内动态成像研究生物大分子或细胞功能的重要工具。本文对现有荧光蛋白的种类和理化特性,及其在生物学研究中的应用进行了综述介绍。重点介绍了近年来荧光蛋白在亮度、Stokes位移、光谱改变等方面的研究进展,介绍了光转换与光活化荧光蛋白及其在超分辨荧光成像技术中的应用。最后对荧光蛋白未来的发展方向进行了展望。     

Fluorescent Biosensors of Intracellular Targets from Genetically Encoded Reporters to Modular Polypeptide Probes

With the escalation of drug discovery programmes, it has become essential to visualize and monitor biological activities in healthy and pathological cells, with high spatial and temporal resolution. To this aim, the development of probes and sensors, which can report on the levels and activities of specific intracellular targets, has become essential. Together with the discovery of the Green Fluorescent Protein (GFP), and the development of GFP-based reporters, recent advances in the synthesis of small molecule fluorescent probes, and the explosion of fluorescence-based imaging technologies, the biosensor field has witnessed a dramatic expansion of fluorescence-based reporters which can be applied to complex biological samples, living cells and tissues to probe protein/protein interactions, conformational changes and posttranslational modifications. Here, we review recent developments in the field of fluorescent biosensor technology. We describe different varieties and categories of fluorescent biosensors together with an overview of the technologies commonly employed to image biosensors in cellulo and in vivo. We discuss issues and strategies related to the choice of synthetic fluorescent probes, labelling, quenching, caging and intracellular delivery of biosensors. Finally, we provide examples of some well-characterized genetically encoded FRET reporter systems, peptide and protein biosensors and describe biosensor applications in a wide variety of fields.     

特异性的蛋白小分子荧光探针及其标记技术

活体蛋白荧光标记技术已经被广泛应用于蛋白质功能的可视化研究中。荧光蛋白常被用来研究蛋白质在生物体内的表达和定位,但由于它本身体积比较大,往往会影响目标蛋白的生物活性。特异性的小分子荧光探针以其体积小、膜透性好、背景噪音低以及制备方便的优点成为蛋白质研究的一个有力工具。本文将简要介绍近几年来各类特异性小分子蛋白荧光探针的研究进展。     

Coassemble dopamine and GHK tripeptide into fluorescent nanoparticles for pH sensing

Fluorescent nanostructures have been widely applied to biomedical researches and clinical diagnosis such as biolabeling/imaging/sensing and have even acted as therapy reagents. Peptide‐based fluorescent nanostructures attract recent interest from biomedical researchers. Inspired by the natural existence of GHK‐Cu complex with a growth factor‐like effect in human blood, here we have developed a novel approach for designing nanosensors through the co‐assembling of two kinds of biomolecules. By making best use of both π‐π stacking between carbon rings and the easy‐oxidation property of an important transmitter molecule, dopamine (DA), we successfully built up a supersensitive and robust fluorescent pH nanosensor by co‐assembling oxidized DA (DA_ox) with a tripeptide GHK. The GHK‐DA_ox nanostructures have a quantum yield of 20.82%, which might be the brightest one among all the current co‐assembling structures merely through unmodified biomolecules. We envision this approach could open a new avenue for not only hybrid nanostructure construction, but also may inspire the bioengineering of in vivo luminescent probes.     

Enzyme-targeted fluorescent small-molecule probes for bacterial imaging

Molecular imaging methods to visualize myriad biochemical processes in bacteria have traditionally been dependent upon molecular biology techniques to incorporate fluorescent biomolecules (e.g., fusion proteins). Such methods have been instrumental in our understanding of how bacteria function but are not without drawbacks, including potential perturbation to native protein expression and function. To overcome these limitations, the use of fluorescent small-molecule probes has gained much attention. Here, we highlight examples from the recent literature that showcase the utility of small-molecule probes for the fluorescence imaging of bacterial cells, including electrophilic, metabolic, and enzyme-activated probes. Although the use of these types of compounds for bacterial imaging is still relatively new, the selected examples demonstrate the exciting potential of these critical tools in the exploration of bacterial physiology.     

In vivo imaging of green fluorescent protein-expressing cells in transgenic animals using fibred confocal fluorescence microscopy

Animal imaging requires the use of reliable long-term fluorescence methods and technology. The application of confocal imaging to in vivo monitoring of transgene expression within internal organs and tissues has been limited by the accessibility to these sites. We aimed to test the feasibility of fibred confocal fluorescence microscopy (FCFM) to image in situ green fluorescent protein (GFP) in cells of living animals. We used transgenic rabbits expressing the enhanced GFP (eGFP) gene. Detailed tissue architecture and cell morphology were visualised and identified in situ by FCFM. Imaging of vasculature by using FCFM revealed a single blood vessel or vasculature network. We also used non-transgenic female rabbits mated with transgenic males to visualise eGFP expression in extra-foetal membranes and the placenta. Expression of the eGFP gene was confirmed by FCFM. This new imaging technology offers specific characteristics: a way to gain access to organs and tissues in vivo, sensitive detection of fluorescent signals, and cellular observations with rapid acquisition at near real time. It allows an accurate visualisation of tissue anatomical structure and cell morphology. FCFM is a promising technology to study biological processes in the natural physiological environment of living animals.     

Occurrence of a bacterial membrane microdomain at the cell division site enriched in phospholipids with polyunsaturated hydrocarbon chains

In this study, we found that phospholipids containing an eicosapentaenyl group form a novel membrane microdomain at the cell division site of a Gram-negative bacterium, Shewanella livingstonensis Ac10, using chemically synthesized fluorescent probes. The occurrence of membrane microdomains in eukaryotes and prokaryotes has been demonstrated with various imaging tools for phospholipids with different polar headgroups. However, few studies have focused on the hydrocarbon chain-dependent localization of membrane-resident phospholipids in vivo. We previously found that lack of eicosapentaenoic acid (EPA), a polyunsaturated fatty acid found at the sn-2 position of glycerophospholipids, causes a defect in cell division after DNA replication of S. livingstonensis Ac10. Here, we synthesized phospholipid probes labeled with a fluorescent 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD) group to study the localization of EPA-containing phospholipids by fluorescence microscopy. A fluorescent probe in which EPA was bound to the glycerol backbone via an ester bond was found to be unsuitable for imaging because EPA was released from the probe by in vivo hydrolysis. To overcome this problem, we synthesized hydrolysis-resistant ether-type phospholipid probes. Using these probes, we found that the fluorescence localized between two nucleoids at the cell center during cell division when the cells were grown in the presence of the eicosapentaenyl group-containing probe (N-NBD-1-oleoyl-2-eicosapentaenyl-sn-glycero-3-phosphoethanolamine), whereas this localization was not observed with the oleyl group-containing control probe (N-NBD-1-oleoyl-2-oleyl-sn-glycero-3-phosphoethanolamine). Thus, phospholipids containing an eicosapentaenyl group are specifically enriched at the cell division site. Formation of a membrane microdomain enriched in EPA-containing phospholipids at the nucleoid occlusion site probably facilitates cell division.     

Imaging mitochondrial reactive oxygen species with fluorescent probes: Current applications and challenges

Abstract

Mitochondrial reactive oxygen species (ROS) is a key element in the regulation of several physiological functions and in the development or progression of multiple pathological events. A key task in the study of mitochondrial ROS is to establish reliable methods for measuring the ROS level in mitochondria with high selectivity, sensitivity, and spatiotemporal resolution. Over the last decade, imaging tools with fluorescent indicators from either small-molecule dyes or genetically encoded probes that can be targeted to mitochondria have been developed, which provide a powerful method to visualize and even quantify mitochondrial ROS level not only in live cells, but also in live animals. These innovative tools that have bestowed exciting new insights in mitochondrial ROS biology have been further promoted with the invention of new techniques in indicator design and fluorescent detection. However, these probes present some limitations in terms of specificity, sensitivity, and kinetics; failure to recognize these limitations often results in inappropriate interpretations of data. This review evaluates the recent advances in mitochondrial ROS imaging approaches with emphasis on their proper application and limitations, and highlights the future perspectives in the development of novel fluorescent probes for visualizing all species of ROS.     

Live-cell imaging with EosFP and other photoactivatable marker proteins of the GFP family

Fluorescent proteins from the green fluorescent protein family have become indispensable imaging tools for cell biology. A wide variety of these proteins were discovered in nonbioluminescent anthozoa in recent years. Some of them feature exciting new properties, with the possibility to alter their intensity and/or fluorescence color by irradiation with light of specific wavelengths. Fluorescent highlighter proteins enable many interesting applications based on regional optical marking in live cells and tissues. This review provides an overview of photoactivatable marker proteins, with a focus on EosFP, a protein that can be switched from green to red fluorescence by approximately 400-nm light. A variety of applications are presented to illustrate the versatility of EosFP in live-cell imaging.     

Live-cell imaging with EosFP and other photoactivatable marker proteins of the GFP family

Fluorescent proteins from the green fluorescent protein family have become indispensable imaging tools for cell biology. A wide variety of these proteins were discovered in nonbioluminescent anthozoa in recent years. Some of them feature exciting new properties, with the possibility to alter their intensity and/or fluorescence color by irradiation with light of specific wavelengths. Fluorescent highlighter proteins enable many interesting applications based on regional optical marking in live cells and tissues. This review provides an overview of photoactivatable marker proteins, with a focus on EosFP, a protein that can be switched from green to red fluorescence by approximately 400-nm light. A variety of applications are presented to illustrate the versatility of EosFP in live-cell imaging.     

Enriched Au nanoclusters with mesoporous silica nanoparticles for improved fluorescence/computed tomography dual‐modal imaging

ObjectivesAu nanoclusters (AuNCs) have been used widely in fluorescence bio‐imaging because of their good fluorescence, small particle size and non‐cytotoxicity. AuNCs are also efficient in computed tomography (CT) imaging. Hence, a dual‐modal imaging probe can be constructed without any complicated modification processes by exploiting the excellent performance of AuNCs. In the present study, AuNCs were enriched with mesoporous silica nanoparticles (MSNs) to obtain enhanced fluorescence/CT dual‐modal imaging, which was capable of acquiring more imaging information for diseases compared with single‐mode imaging.Materials and methodsBiocompatible bovine serum albumin (BSA)‐capped AuNCs were prepared and loaded into amine‐functionalized MSNs to form MSN@AuNCs. BSA‐AuNCs, MSNs, and MSN@AuNCs were characterized by ultraviolet‐visible (UV‐vis) spectra, transmission electron microscopy (TEM), fluorescence spectra, and zeta potential. CT imaging was recorded using micro‐CT scanning. Fluorescence imaging was measured using confocal laser scanning microscopy and flow cytometry.ResultsThe prepared AuNCs and MSNs possessed good properties as previously reported. The fluorescence intensity and CT value of the AuNCs were enhanced after being enriched with MSNs. The nanoparticles were both non‐cytotoxic. Confocal laser scanning microscopy and flow cytometry indicated that MSN@AuNCs in CAL‐27 cells showed improved fluorescence imaging compared with simple AuNCs at the same concentration.ConclusionsThe results revealed that the strategy of enriching AuNCs with MSNs can obtain highly sensitive fluorescence/CT dual‐modal imaging, which indicated the potential of this nanoparticle in the diagnosis and treatment of disease.     

基于受激发射损耗显微术的活细胞和活体超分辨成像

细胞作为生命体基本的结构和功能单元，在生物、医学等领域有着非常重要的研究意义。随着现代科学和技术的发展，科学家们借助电镜对细胞以及细胞器的空间结构已经有非常清晰的认识，但是对它们的功能以及细胞之间的相互作用却了解得非常少，而这恰恰又是疾病治疗和药物开发亟需了解的信息，因此对离体活细胞（简称活细胞）和活体生物组织细胞（简称活体细胞）中亚细胞器的研究变得非常重要。然而细胞中许多细胞器的结构在纳米量级，传统的光学成像技术由于受到光学衍射极限的限制是无法观察到纳米量级的生物结构，因此光学超分辨成像技术是目前研究亚细胞器结构和功能的有效工具。在所有光学超分辨显微技术中，受激发射损耗显微术（stimulated emission depletionmicroscopy,STED）由于具有实时成像、三维超分辨和断层成像的能力，非常适合用于纳米尺度的活细胞和活体细胞成像研究，而且STED超分辨成像技术经过近几十年的发展，已经广泛用于活细胞甚至活体小鼠细胞的超分辨动态观测。本文总结了近年来活细胞和活体小鼠神经元细胞等领域STED超分辨成像的研究进展，介绍了用于活细胞和活体细胞STED超分辨成像的荧光染料...     

Potentials and pitfalls of fluorescent quantum dots for biological imaging

Fluorescent semiconductor nanocrystals, known as quantum dots (QDs), have several unique optical and chemical features. These features make them desirable fluorescent tags for cell and developmental biological applications that require long-term, multi-target and highly sensitive imaging. The improved synthesis of water-stable QDs, the development of approaches to label cells efficiently with QDs, and improvements in conjugating QDs to specific biomolecules have triggered the recent explosion in their use in biological imaging. Although there have been many successes in using QDs for biological applications, limitations remain that must be overcome before these powerful tools can be used routinely by biologists.     

Recent advances using green and red fluorescent protein variants

Fluorescent proteins have proven to be excellent tools for live-cell imaging. In addition to green fluorescent protein (GFP) and its variants, recent progress has led to the development of monomeric red fluorescent proteins (mRFPs) that show improved properties with respect to maturation, brightness, and the monomeric state. This review considers green and red spectral variants, their paired use for live-cell imaging in vivo, in vitro, and in fluorescence resonance energy transfer (FRET) studies, in addition to other recent “two-color” advances including photoswitching and bimolecular fluorescence complementation (BiFC). It will be seen that green and red fluorescent proteins now exist with nearly ideal properties for dual-color microscopy and FRET.