Multicolor Bioimaging Research Articles

Naphthalimide-sulfonamide fused dansyl-sulfonamide fluorescent probe for tracking glutathione of lysosome with a dual-emission manner

Developing glutathione-specific fluorescent probes with novel fluorescence-responsive manners can enrich the applications of probes in living cells. In this work, we designed and synthesized a dual fluorophores (naphthalimide and dansyl) co-modified fluorescent probe, which displayed a high selectivity toward glutathione (GSH). In living cells, this probe could specifically visualize the GSH of lysosome and accompanied by two different fluorescence emissions. One is green emission that can be assigned to the nucleophilic substitution product (GSH-linked naphthalimide) while the other is orange emission that can be derived from the cleavage product (dansyl). Especially, such dual-emission bioimaging in living cells had a spatiotemporal and synchronous manner. It is expected to provide an alternative strategy for designing the functional fluorescent probes to be applied in multi-color bioimaging in future.

A Large Stokes Shift Fluorescent Protein Constructed from the Fusion of Red Fluorescent mCherry and Far-Red Fluorescent BDFP1.6.

Phycobiliproteins are constituents of phycobilisomes that can harvest orange, red, and far-red light for photosynthesis in cyanobacteria and red algae. Phycobiliproteins in the phycobilisome cores, such as allophycocyanins, absorb far-red light to funnel energy to the reaction centers. Therefore, allophycocyanin subunits have been engineered as far-red fluorescent proteins, such as BDFP1.6. However, most current fluorescent probes have small Stokes shifts, which limit their applications in multicolor bioimaging. mCherry is an excellent fluorescent protein that has maximal emittance in the red spectral range and a high fluorescence quantum yield, and thus, can be used as a donor for energy transfer to a far-red acceptor, such as BDFP1.6, by FRET. In this study, mCherry was fused with BDFP1.6, which resulted in a highly bright far-red fluorescent protein, BDFP2.0, with a large Stokes shift (≈79 nm). The excitation energy was absorbed maximally at 587 nm by mCherry and transferred to BDFP1.6 efficiently; thus emitting strong far-red fluorescence maximally at 666 nm. The effective brightness of BDFP2.0 in mammalian cells was 4.2-fold higher than that of iRFP670, which has been reported as the brightest far-red fluorescent protein. The large Stokes shift of BDFP2.0 facilitates multicolor bioimaging. Therefore, BDFP2.0 not only biolabels mammalian cells, including human cells, but also biolabels various intracellular components in dual-color imaging.

SyncRGB-FLIM: synchronous fluorescence imaging of red, green and blue dyes enabled by ultra-broadband few-cycle laser excitation and fluorescence lifetime detection.

We demonstrate for the first time that an ultra-broadband 7 femtosecond (fs) few-cycle laser can be used for multicolor nonlinear imaging in a single channel detection geometry, when employing a time-resolved fluorescence detection scheme. On a multi-chromophore-labelled cell sample we show that the few-cycle laser can efficiently excite the multiple chromophores over a >400 nm two-photon absorption range. By combining the few-cycle laser excitation with time-correlated single-photon counting (TCSPC) detection to record two-photon fluorescence lifetime imaging microscopy (FLIM) images, the localization of different chromophores in the cell can be identified based on their fluorescence decay properties. The novel SyncRGB-FLIM multi-color bioimaging technique opens the possibility of real-time protein-protein interaction studies, where its single-scan operation translates into reduced laser exposure of the sample, resulting in more photoprotective conditions for biological specimens.

Multi-colored type-I Ag-doped ZnCdS/ZnS core/shell quantum dots with intense emission

Abstract Although doped quantum dots (d-dots) with intense and tunable emission have been studied for long time, there are still great efforts to prepare new ones with promising characteristics. In the present work, we used a mild/effective strategy for preparing high quality and aqueous-soluble Ag:ZnCdS/ZnS core/shell quantum dots (QDs) with N-acetyl- l -cysteine as the capping agent. Through investigating the experimental variables, the impurity-related emission intensity of the as-prepared samples was optimized. The capability of the present work on creating quantum structures with tunable emission across the entire visible spectrum was approved through simple but effective variation in Zn-to-Cd molar ratio. Indeed, as the Zn:Cd molar ratio changed from 2:0 to 0:2, the emission color was changed significantly from blue to red color with a satisfactory photoluminescence quantum yield. The quantum yield value reached ∼41% for the as-prepared core/shell d-dots without any pre/post-treatment, which is a remarkable result for such aqueous-soluble structures. XRD, EDX, ICP, and TEM measurements were applied to determine the structural features of the QDs in a strong quantum confinement regime. The generality of preparation route, its biocompatibility, along with a multi-color emission, can create new opportunities especially for the white light emitting technologies, or multi-color bioimaging for theranostics.

Full-color carbon dots with multiple red-emission tuning: on/off sensors, in vitro and in vivo multicolor bioimaging

Carbon-based luminescent materials have attracted much attention of many researchers in recent years for its better optical properties and biological applications. In this paper, we report the full-color CDs (from blue to red) with better quality red emissive by a facile and efficient synthesis route. The CDs show multiple red-emission tuning, unique nature of up-conversion photoluminescent under lower excitation and single-particle photoluminescent. The luminescence mechanism was also explored preliminarily. Especially, the results demonstrate that full-color CDs with multiple red wavelength tuning were achieved successfully by using only one precursor and this phenomenon was reasonably explored by using solvatochromism mechanism instead of only excitation-dependent or emission tuning. Moreover, the multicolor FL emission of CDs is caused by single particles rather than a mixture with diverse photoluminescent, which can be expanded to precision manufacture field on luminance, but not just limited to multidimensions biosensing. In addition, UCPL under lower excitation implies the more important potential applications in surface layer bioimaging, microarray detection and light activation technology in vivo and in vitro. The full-color CDs show high quantum yield (73.89%) and superior anti-photobleaching. Multicolor bioimaging, in vitro and in vivo, indicates its low toxicity in Hela cells and zebrafish. Besides, the as-prepared CDs were successfully used for multidimensions sensing, as Fe3+ metal ion sensing (on/off).

Indian Gooseberry-Derived Tunable Fluorescent Carbon Dots as a Promise for In Vitro/In Vivo Multicolor Bioimaging and Fluorescent Ink

We report the synthesis of eco-friendly fluorescent nitrogen-doped carbon dots (NCDs) using the renewable resource of Phyllanthus emblica juice as a precursor by the hydrothermal process at 200 °C ...

Preparation of carbon dots with long-wavelength and photoluminescence‐tunable emission to achieve multicolor imaging in cells

Most carbon dots (CDs) derived from natural biowastes show emission limitedly in the blue range. This work applied urea and sulfuric acid as dopants to assist the carbonization of cellulose-based biowaste willow catkin. Then a facile chemical cutting method was developed for scalable production of CDs. Under excitation at a single-wavelength of 360 nm, the aqueous solutions of CDs obtained at different cutting temperatures (100, 120, and 140 °C) exhibited strong blue (486 nm), green (522 nm) and yellow (545 nm) emission, respectively. The as-prepared CDs also exhibited excitation-independent emission behaviors and high photostability in different pH and ionic strength. Furthermore, these long-wavelength and photoluminescence-tunable CDs were successfully applied to multicolor bioimaging in Hela cells, indicating their potential toward multimodal sensing applications in biological environment.

Bioluminescent imaging: new opportunities

Modern biomedical research technologies actively use bioimaging for studying cells, tissues and whole organisms. Multicolor bioimaging is applied when simultaneous observation of different events at the molecular and cellular level is needed. Bioluminescent imaging methods are the most sensitive, however, their use for multicolor labeling is complicated due to the insufficient number of available uciferin-luciferase pairs. Having a number of advantages compared to previously studied bioluminescent systems, the new bioluminescence systems of higher fungi and marine polychaete Odontosyllis could become a useful expansion of the bioimaging toolbox.

Covalently Assembled Dipeptide Nanoparticles with Adjustable Fluorescence Emission for Multicolor Bioimaging.

Peptide self-assembly, inspired by the naturally occurring fabrication principle, remains the most attractive in constructing fluorescent nanoagents towards bioimaging. However, the noncovalent interactions that drive peptide self-assembly are usually susceptible to the complex physiological environment; thus leading to disassembly and dysfunction of fluorescent nanoagents. Herein, a covalently crosslinked assembly strategy for fabrication of stable peptide-based nanoparticles with adjustable emission is introduced. In the process of cationic diphenylalanine peptide (H-Phe-Phe-NH2 ⋅HCl) self-assembly, glutaraldehyde is used as a crosslinker and the resulting product of the Schiff base reaction can be fluorescent. More importantly, the emission wavelength can be readily tuned by controlling the covalent reaction time. It has been demonstrated that the nanoparticles are stable, even after intracellular uptake, and can be used for sustainable multicolor fluorescent imaging. The strategy with integrating peptide self-assembly and covalent crosslinking could be promising for the design and engineering of functional fluorescent nanoparticles with robust physiological stability and adjustable emission towards improved bioimaging applications.

Quick Microwave Assisted Synthesis and In Vitro Imaging Application of Oxygen Doped Fluorescent Carbon Dots.

In this paper, a fast and simplest one-pot tactic was used to synthesis fluorescent oxygen doped carbon dots from Tween-20 (TTO-CDs) is reported. The TTO-CDs were microwavically synthesized by using Tween-20 as both the carbon precursor and the oxygen dopant as well. The surface morphology, crystalline and/or amorphous nature, composition and optical assets of synthesized TTO-CDs were studied by means of existing techniques. From the results, it was confirmed that the as-synthesized TTO-CDs are amorphous in nature, monodispersed, sphere-shaped and the typical particle size range is 5 ± 1.5nm. The synthesized TTO-CDs emits strong blue fluorescence at 390nm under excitation of 335nm. Most interestingly, the excitation dependent emission property of synthesized TTO-CDs was exposed from fluorescence results. The synthesized TTO-CDs have quantum yield of about 14% against quinine sulfate as reference standard. The biotoxicity of synthesized TTO-CDs on HeLa cells was assessed through cytotoxicity assay. These results implied that the fluorescent TTO-CDs showed less biotoxicity, and further which was efficaciously applied as a multicolor staining and bioimaging probe for the confocal imaging of HeLa cells.

Reversible "Off-On" Fluorescence of Zn2+-Passivated Carbon Dots: Mechanism and Potential for the Detection of EDTA and Zn2.

Zn2+-passivated carbon dots (named Z-CDs) were synthesized from zinc gluconate for the first time through a one-step pyrolysis treatment. The mechanism of Zn2+-enhanced fluorescence was carefully investigated, and a new strategy to passivate the surfaces of CDs by Zn2+ was proposed. Inspired by the complexation reaction between Zn2+ and ethylenediaminetetraacetic acid (EDTA), a reversible "off-on" fluorescent nanosensor for the detection of EDTA and Zn2+ was constructed based on the depassivation and repassivation of Z-CDs, with a limit of detection as low as 3.2 × 10-7 M and 5.1 × 10-7 M, respectively. The proposed Z-CD-based nanosensor had been further utilized for EDTA and Zn2+ monitoring in tap water with excellent recovery. To the best of our knowledge, this was the first report of a fluorescence-based sensor of EDTA and a turn-on sensor of Zn2+ based on CDs with reversible detection capability. Also, benefiting from the low toxicity of zinc, Z-CDs were applied for multicolor bioimaging and in vitro detection in cells.

An Unconventional Polymerization Route to Hydrophilic Fluorescent Organic Nanoparticles for Multicolor Cellular Bioimaging

We demonstrated an unconventional polymerization route to synthesize hydrophilic fluorescent organic nanoparticles (FONs) for multicolor cellular bioimaging in this contribution. The route benefits from our unexpected discovery of a rapid polymerization reaction between 1,6-hexanediol dipropiolate and 2,4,6-triazide-1,3,5-triazine under the catalysis of N,N,N',N'',N''-pentamethyldiethylenetriamine (PMDETA). Interestingly, the 2,4,6-triazide-1,3,5-triazine and PMDETA system can also induce rapid free radical polymerization at room temperature. The as-prepared FONs exhibited promising water solubility and stability with an average diameter of 20 nm. The excitation wavelength-dependent fluorescent properties endow the FONs with blue, yellow, and red fluorescent emission under UV, blue, and green excitation, respectively. The cytotoxicity of FONs was investigated by using a Cell Counting Kit (CCK-8) assay, which indicated good biocompatiblity. More importantly, the cell uptake experiment verified the FONs were excellent fluorescent nanoprobes for multicolor cellular bioimaging. Therefore, this unconventional route provides a novel fabrication strategy of highly hydrophilic FONs for biomedical applications.

Aqueous synthesis of L-cysteine and mercaptopropionic acid co-capped ZnS quantum dots with dual emissions

In this paper, L-cysteine (L-cys) and mercaptopropionic acid (MPA) co-capped ZnS quantum dots (QDs) with dual emissions have been successfully synthesized by a one-pot aqueous-phase synthesis method. The intensities of the dual emissions could be controlled by regulating the molar ratio of L-cys to MPA, and the fluorescence color also turned from blue to yellow accordingly. The relationship between the ligands and fluorescence was investigated and the results indicated that L-cys could cause two emissions and MPA improved the emission intensity. In addition, the L-cys-MPA co-capped ZnS QDs showed high photostability under UV irradiation. Therefore, the L-cys-MPA co-capped ZnS QDs, which show the dual emissions and tunable emission intensities, have great potentials for use in ratiometric fluorescence sensors and multicolor bioimaging.

Atomistic tight-binding simulations of quaternary-alloyed $$\hbox {Zn}_{x}\hbox {Cd}_{1-x}\hbox {S}_{y}\hbox {Se}_{1-y}$$ Zn x Cd 1 - x S y Se 1 - y nanocrystals

Advancement of alloyed nanocrystals with attractive structural and optical properties for use in a wide range of physical, chemical, and biological applications represents a growing research field. Employing atomistic tight-binding theory combined with the virtual crystal approximation, the electronic structure and optical properties of quaternary-alloyed $$\hbox {Zn}_{{x}}\hbox {Cd}_{1-{x}} \hbox {S}_{{y}}\hbox {Se}_{1-{y}}$$ nanocrystals with experimentally synthesized compositions (x and y) and sizes were investigated. Analysis of the results shows that the physical properties are mainly sensitive to the concentrations (x and y) and the diameter. With decreasing x and y contents, the optical bandgap is reduced because the contributions of the materials with narrower bulk bandgap (ZnSe and CdSe) is mostly promoted. The optical bandgap is reduced with increasing diameter due to the quantum confinement effect. The optical bandgap calculated based on tight-binding calculations shows discrepancy of less than 0.4 eV from experiment. Most importantly, the optical emission is continuously tunable across the entire visible spectrum. The conduction and valence bands are predominantly contributed by cation and anion atoms, respectively. The optical properties are obviously improved in Cd- and Se-rich quaternary $$\hbox {Zn}_{{x}}\hbox {Cd}_{1-{x}} \hbox {S}_{{y}}\hbox {Se}_{1-{y}}$$ nanocrystals with large diameter. The atomistic electron–hole interactions can be hybrid-engineered by tuning either the contents (x and y) or diameter. The Stokes shift becomes more pronounced with decreasing alloy concentrations (x and y) and diameter, as described by the trend of the atomistic electron–hole exchange interaction. The present systematic study provides a new avenue to understand the unique size- and composition-dependent structural and optical properties of quaternary-alloyed $$\hbox {Zn}_{{x}}\hbox {Cd}_{1-{x}} \hbox {S}_{{y}}\hbox {Se}_{1-{y}}$$ nanocrystals for broad use in multicolor bioimaging, biosensing, light-emitting diodes, solar cells, and other nanodevice applications.

Quick synthesis of 2-propanol derived fluorescent carbon dots for bioimaging applications

Herein, for the first time, we present a one-pot ingenious preparative method for fluorescent carbon dots from 2-propanol (2P-CDs) without external treatments. Structure, morphology, chemical composition and fluorescence properties of the 2P-CDs were examined. These results confirm that the as-synthesized 2P-CDs are amorphous, monodispersed, spherical and the average particle size is 2.5 ± 0.7 nm. Most importantly, excitation-dependent emission properties were observed, which suggest that these 2P-CDs may be used in multicolor bioimaging applications. When incubated with HeLa cells, the 2P-CDs exhibit low cytotoxicity, and positive biocompatibility. Confocal microscopy image shows the uptake of 2P-CDs by HeLa cells and the application of probable biomarker is demonstrated.

Concentration-dependent color tunability of nitrogen-doped carbon dots and their application for iron(III) detection and multicolor bioimaging

Nitrogen doping can effectly adjust the compositions and structures of carbon dots and hence enhance their fluorescence. In this work, we report a fast and low-cost route for synthesis of nitrogen-doped carbon dots (N-CDs) by microwave pyrolysis of citric acid and ammonium within 7 min. The as-prepared N-CDs contain plentiful oxygen and nitrogen functional groups, and dispaly intense fluorescence with high quantum yield of ca. 44.3% and have an average size of 1.8 nm. The obtained N-CDs exhibit highly stable against photobleaching, ionic strengths, and can be used for selective and sensitive detection of Fe(III). It is postulated that the Fe3+-mediated fluorescence quenching is attributed to the charge transfer between N-CDs and Fe3+. In particular, the emission peaks from blue to red region can be tuned by interparticle distance of N-CDs, simply by increasing the concentration of N-CDs in aqueous solution, which indicates its potential applications as a promising optical image probe in multicolor cellular imaging.

Versatile multicolor nanodiamond probes for intracellular imaging and targeted labeling.

We report on the sizable production of fluorescent nanodiamonds (FNDs) containing a near infrared (NIR) color center - namely the silicon vacancy (SiV) defect, and their first demonstration inside cells for bio-imaging. We further demonstrate a concept of multi-color bio-imaging using FNDs to investigate intercellular processes using two types of FNDs. Due to their specific spectral properties, SiV FNDs can be distinguished from common nitrogen-vacancy (NV) FNDs and show a distinct initial spreading throughout the cell interior. The reported results are the first demonstration of multi-color labeling with FNDs that are especially interesting for in vivo bio-imaging due to their stable fluorescence.

Highly photoluminescent N-isopropylacrylamide (NIPAAM) passivated carbon dots for multicolor bioimaging applications

We report a highly luminescent Carbon Dots passivated by nitrogen rich molecules, i.e. N-isopropylacrylamide (NIPAAM), with a QY as high as ca. 94%, which is the highest yield until now. NIPAMM@C-Dots are synthesized directly from the precursors composed of CA, EDA and NIPAAM by hydrothermal reaction. The optical properties of in situ synthesized NIPAAM@C-Dots showed EX-dependent EM at high concentration and EX-independent EM at low concentration. In most cations and anions except Fe3+ and Bi3+ added to the aqueous solution, NIPAAM@C-Dots exhibit an extremely stable inert behavior. In addition, in order to verify its usefulness as a fluorescent marker, C6 glioblastoma cells and NIPAAM@C-Dots were cultured together and then applied by both confocal bioimaging and flow cytometry analysis. The role of NIPAAM present on or around the C-Dots surface can improve QY values and stability, resulting in reduced cytotoxicity and can be a strong candidate as an alternative to organic dyes.

Dual-Color-Emitting Carbon Nanodots for Multicolor Bioimaging and Optogenetic Control of Ion Channels.

The development of intrinsically multicolor‐emitting carbon nanodots (CNDs) has been one of the great challenges for their various fields of applications. Here, the controlled electronic structure engineering of CNDs is performed to emit two distinct colors via the facile surface modification with 4‐octyloxyaniline. The so‐called dual‐color‐emitting CNDs (DC‐CNDs) can be stably encapsulated within poly(styrene‐co‐maleic anhydride) (PSMA). The prepared water‐soluble DC‐CNDs@PSMA can be successfully applied to in vitro and in vivo dual‐color bioimaging and optogenetics. In vivo optical imaging can visualize the biodistribution of intravenously injected DC‐CNDs@PSMA. In addition, the light‐triggered activation of ion channel, channelrhodopsin‐2, for optogenetic applications is demonstrated. As a new type of fluorophore, DC‐CNDs offer a big insight into the design of charge‐transfer complexes for various optical and biomedical applications.

π-Conjugated Chromophore Incorporated Polystyrene Nanobeads as Single Optical Agent for Three-Channel Fluorescent Probe in Bioimaging Application.

Fluorescent polystyrene (PS) nanobeads in the size range ∼70-120 nm incorporating perylene bisimide (PBI-PS) and/or oligo(p-phenylenevinylene) (OPV-PS) was developed by miniemulsion polymerization technique. A dye loading content (DLC) of <3% was sufficient to impart high fluorescence emission capability to the PS beads. OPV-PS exhibited emission in the range 400-550 nm with peak emission at 450 nm (λex = 350 nm; ϕFL = 26%); PBI-PS showed emission from 520-650 nm with peak emission at 545 nm (λex = 490 nm; ϕFL = 9.7%) in 1× PBS buffer, whereas OPV(PBI)-PS nanobeads incorporating both the fluorophores exhibited multicolor emission capabilities (λex from 350 to 490 nm). The nanoparticles were characterized by field-emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM) and dynamic light scattering (DLS) for size and zeta potential for surface charge. For bioimaging applications, the PS nanoparticles were incubated with HeLa cells. Cell viability analysis involving HeLa cells showed more than 90% cell viability confirming the biocompatibility of the PS beads. The cellular uptake of the nanoparticles was confirmed by flow cytometry analysis and confocal laser scanning microscopy (CLSM) images. The subcellular localization of the nanoparticles in the cytoplasm could be precisely established by their simultaneous multicolor emission. The PS-based single optical agent presented here that can function as three-channel fluorescent probe to meet the requirements for multicolor bioimaging is advantageous.