Multicolor Live Cell Imaging Research Articles

Multi-color live-cell fluorescence imaging of primary ciliary membrane assembly and dynamics.

The ciliary membrane is continuous with the plasma membrane but has distinct lipid and protein composition, which is key to defining the function of the primary cilium. Ciliary membranes dynamically assemble and disassemble in association with the cell cycle and directly transmit signals and molecules through budding membranes. Various imaging approaches have greatly advanced the understanding of the ciliary membrane function. In particular, fluorescence live-cell imaging has revealed important insights into the dynamics of ciliary membrane assembly by monitoring the changes of fluorescent-tagged ciliary proteins. Protein dynamics can be tracked simultaneously using multi-color live cell imaging by coupling ciliary-associated factors with different colored fluorescent tags. Ciliary membrane and membrane associated-proteins such as Smoothened, 5-HTr6, SSTR3, Rab8a, and Arl13b have been used to track ciliary membranes and centriole proteins like Centrin1/2, CEP164, and CEP83 are often used to mark the ciliary basal body. Here, we describe a method for studying ciliogenesis membrane dynamics using spinning disk confocal live-cell imaging.

Acidic pH-Triggered Live-Cell Lysosome Specific Tracking, Ratiometric pH Sensing, and Multicolor Imaging by Visible to NIR Switchable Cy-7 Dyes.

We have demonstrated an efficient synthetic route with crystal structures for the construction of acidic pH-triggered visible-to-NIR interchangeable ratiometric fluorescent pH sensors. This bioresponsive probe exhibits pH-sensitive reversible absorption/emission features, low cytotoxicity, a huge 322 nm bathochromic spectral shift with augmented quantum yield from neutral to acidic pH, high sensitivity and selective targeting ability of live-cell lysosomes with ideal pKa , off-to-on narrow NIR absorption/fluorescence signals with high molar absorption coefficient at acidic lysosomal lumen, and in-situ live-cell pH-activated ratiometric imaging of lysosomal pH. Selective staining and ratiometric pH imaging in human carcinoma live-cell lysosomes were monitored by dual-channel confocal laser scanning microscope using a pH-activatable organic fluorescent dye comprising a morpholine moiety for lysosome targeting and an acidic pH openable oxazolidine ring. Moreover, real-time tracking of lysosomes, 3D, and multicolor live-cell imaging have been achieved using the synthesized pH-activatable probe.

Super-resolved live-cell imaging using random illumination microscopy

SummaryCurrent super-resolution microscopy (SRM) methods suffer from an intrinsic complexity that might curtail their routine use in cell biology. We describe here random illumination microscopy (RIM) for live-cell imaging at super-resolutions matching that of 3D structured illumination microscopy, in a robust fashion. Based on speckled illumination and statistical image reconstruction, easy to implement and user-friendly, RIM is unaffected by optical aberrations on the excitation side, linear to brightness, and compatible with multicolor live-cell imaging over extended periods of time. We illustrate the potential of RIM on diverse biological applications, from the mobility of proliferating cell nuclear antigen (PCNA) in U2OS cells and kinetochore dynamics in mitotic S. pombe cells to the 3D motion of myosin minifilaments deep inside Drosophila tissues. RIM's inherent simplicity and extended biological applicability, particularly for imaging at increased depths, could help make SRM accessible to biology laboratories.

Fluorescent-Nitrogen-Doped Carbon Quantum Dots Derived from Citrus Lemon Juice: Green Synthesis, Mercury(II) Ion Sensing, and Live Cell Imaging.

In this study, we report a green and economical hydrothermal synthesis of fluorescent-nitrogen-doped carbon quantum dots (NCQDs) using citrus lemon as a carbon source. The prepared NCQDs possess high water solubility, high ionic stability, resistance to photobleaching, and bright blue color under ultraviolet radiation with a high quantum yield (∼31%). High-resolution transmission electron microscopy (HRTEM) results show that the prepared NCQDs have a narrow size distribution (1–6 nm) with an average particle size of 3 nm. The mercury ion (Hg2+) sensing efficiency of the NCQDs was studied, and the result indicated that the material has high sensitivity, high precision, and good selectivity for Hg2+. The limit of detection (LOD) is 5.3 nM and the limit of quantification (LOQ) is 18.3 nM at a 99% confidence level. The cytotoxicity was evaluated using MCF7 cells, and the cell viabilities were determined to be greater than 88% upon the addition of NCQDs over a wide concentration range from 0 to 2 mg/mL. Based on the low cytotoxicity, good biocompatibility, and other revealed interesting merits, we also applied the prepared NCQDs as an effective fluorescent probe for multicolor live cell imaging.

Photoluminescence properties of gadolinium phosphate nanoprisms doped with lanthanide ions for multicolor live cell imaging

Lanthanide fluorescence nanoprobe is versatile in multicolor cell imaging, although it still faces a great challenge in improving its luminescent efficiency. Herein, we validated the feasibility of lanthanide ions co-doped gadolinium phosphate nanocrystals serving as a safe, effective and color-tunable fluorescence probe for live cell imaging. Well-crystallized hexagonal phase GdPO4 nanoparticles with multi-morphologies such as nanowires, nanorods, tetragonal nanocubes and hexagonal nanoprisms were synthesized via a typical hydrothermal method. We have systematically investigated the synthesis of different morphologies of GdPO4 nanoparticles through adjusting their processing parameters. The nanoparticles can gain a bright color-tunable photoluminescence from red, orange, yellow to green region when the GdPO4 nanoparticles are co-doped with Eu3+ and Tb3+ ions. The Tb3+ doping significantly enhances the characteristic 4f → 4f transitions of Eu3+ owing to a specific cross-relaxation process from Tb3+ to Eu3+ ions; the mechanism underlying the cross relaxation was exposited in detail. Further biological experiments in vitro proved that the prepared nanoprisms could exhibit a favourable biocompatibility with negligible toxicity. The current investigation manifests a high potential of the nanoparticles applicable to fluorescence imaging probe for live cells.

“Vigna radiata” based green C-dots: Photo-triggered theranostics, fluorescent sensor for extracellular and intracellular iron (III) and multicolor live cell imaging probe

Fluorescent C-dots have drawn increasing attention due to their superior properties such as high aqueous solubility, optical absorptivity, favorable biocompatibility and appreciable photoluminescence. Nature offers various precursors for the synthesis of green C-dots like fruits, vegetables, flowers, animal derivatives and many more. Herein, nitrogen doped C-dots (N@VRCD) were developed by economical, one step hydrothermal method using “Vigna radiata” sprouts as sole carbon precursor and ethylenediamine as the nitrogen source, which was optimized for enhanced optical properties. To the best of our knowledge, N@VRCD shows excellent quantum yield ∼58%. Importantly, N@VRCD also exhibits significant light induced phototoxicity to cancerous cells which enable the possibility of using it as a potential theranostics agent. Further, the N@VRCD displays highly sensitive and selective sensing behaviour towards Fe3+ ions by getting fluorescence “turn-off” with the limit of detection as low as 140 nM. Moreover, N@VRCD shows biocompatible and hemocompatible nature with a potential of multicolor live cell imaging and intracellular Fe3+ detection which was validated by confocal microscopy and flow cytometry.

Full color emitting fluorescent carbon material as reversible pH sensor with multicolor live cell imaging

Carbon-based nano materials are developed as a cytocompatible alternative to semiconducting quantum dots for bioimaging and fluorescence-based sensing. The green alternatives for the synthesis of carbon materials are imminent. The present study demonstrates microwave based one step quick synthesis of fluorescent carbon material (FCM) having three variants: (i) un-doped fluorescent carbon material (UFCM) (ii) nitrogen doped FCM (N@FCM), and (iii) nitrogen & phosphorus co-doped FCM (N-P@FCM) using sugarcane extract as a carbon source. The N doping was performed using ethylenediamine and phosphoric acid was used for P doping. The heteroatom doped FCM were synthesized due to insolubility of UFCM in water. Unlike, UFCM, the N@FCM and N-P@FCM were found to be highly soluble in water. The N-P@FCM shows highest quantum yield among the three. The N-P@FCM was explored for alkaline pH sensing and it shows a quenching of fluorescence in the pH range 09–14. The sensing behaviour shows reversibility and high selectivity. Further, the sensor was also investigated for their biocompatibility and hence employed as a promising multicolour probe for cancer cell imaging. The generality in cell imaging was investigated by flow cytometry. The hetero-atom doped green carbon-dots may open new avenues for sensing and selective cellular targeting.

Staining of Lipid Droplets with Monodansylpentane.

Monodansylpentane (MDH) is a fluorophore that displays selective labeling of lipid droplets (LDs). The dye preferentially segregates into the neutral lipid cores of LDs and emits blue fluorescence, compatible with the simultaneous use of green and red fluorescent reporters in multi-color live-cell imaging. MDH can be used for visualizing LDs not only in cell cultures, but also in fixed tissues such as the fat body and ovaries from Drosophila. MDH is therefore a versatile marker for LDs in fluorescence microscopy.

HIV-1 and M-PMV RNA Nuclear Export Elements Program Viral Genomes for Distinct Cytoplasmic Trafficking Behaviors.

Retroviruses encode cis-acting RNA nuclear export elements that override nuclear retention of intron-containing viral mRNAs including the full-length, unspliced genomic RNAs (gRNAs) packaged into assembling virions. The HIV-1 Rev-response element (RRE) recruits the cellular nuclear export receptor CRM1 (also known as exportin-1/XPO1) using the viral protein Rev, while simple retroviruses encode constitutive transport elements (CTEs) that directly recruit components of the NXF1(Tap)/NXT1(p15) mRNA nuclear export machinery. How gRNA nuclear export is linked to trafficking machineries in the cytoplasm upstream of virus particle assembly is unknown. Here we used long-term (>24 h), multicolor live cell imaging to directly visualize HIV-1 gRNA nuclear export, translation, cytoplasmic trafficking, and virus particle production in single cells. We show that the HIV-1 RRE regulates unique, en masse, Rev- and CRM1-dependent “burst-like” transitions of mRNAs from the nucleus to flood the cytoplasm in a non-localized fashion. By contrast, the CTE derived from Mason-Pfizer monkey virus (M-PMV) links gRNAs to microtubules in the cytoplasm, driving them to cluster markedly to the centrosome that forms the pericentriolar core of the microtubule-organizing center (MTOC). Adding each export element to selected heterologous mRNAs was sufficient to confer each distinct export behavior, as was directing Rev/CRM1 or NXF1/NXT1 transport modules to mRNAs using a site-specific RNA tethering strategy. Moreover, multiple CTEs per transcript enhanced MTOC targeting, suggesting that a cooperative mechanism links NXF1/NXT1 to microtubules. Combined, these results reveal striking, unexpected features of retroviral gRNA nucleocytoplasmic transport and demonstrate roles for mRNA export elements that extend beyond nuclear pores to impact gRNA distribution in the cytoplasm.

Characterization of Fluorescent Proteins for Three- and Four-Color Live-Cell Imaging in S. cerevisiae.

Saccharomyces cerevisiae are widely used for imaging fluorescently tagged protein fusions. Fluorescent proteins can easily be inserted into yeast genes at their chromosomal locus, by homologous recombination, for expression of tagged proteins at endogenous levels. This is especially useful for incorporation of multiple fluorescent protein fusions into a single strain, which can be challenging in organisms where genetic manipulation is more complex. However, the availability of optimal fluorescent protein combinations for 3-color imaging is limited. Here, we have characterized a combination of fluorescent proteins, mTFP1/mCitrine/mCherry for multicolor live cell imaging in S. cerevisiae. This combination can be used with conventional blue dyes, such as DAPI, for potential four-color live cell imaging.

Green preparation of carbon dots for intracellular pH sensing and multicolor live cell imaging.

Carbon dots are prepared using a green hydrothermal approach with dehydrated shiitake mushroom as the sole carbon source without any additives (these carbon dots are shortly termed as MCDs). Carbonization, surface functionalization and nitrogen doping are involved in the hydrothermal treatment and no further modification or surface passivation is necessary. The derived MCDs are nitrogen-doped, oxygen-rich with hydroxyl, carboxyl and amine groups, with a diameter of ca. 4.2 nm. MCDs exhibit cell permeable properties and a distinct pH-sensitive/excitation-dependent photoluminescence emission feature within pH 4.0-8.0, providing an optical probe for intracellular pH sensing and multicolor imaging of live HeLa cells. MCDs also show a strong fluorescence response to hemin, which facilitates sensitive fluorescent sensing of hemin with a detection limit of 120 nmol L-1.

Triggering mitophagy with far-red fluorescent photosensitizers.

Cells identify defective mitochondria and eliminate them through mitophagy: this allows cells to rid themselves of unwanted stress to maintain health and avoid the activation of cell death. One approach to experimentally investigate mitophagy is through the use of mitochondrial photosensitizers, which when coupled with light allows one to precisely control mitochondrial damage with spatial and temporal precision. Here we report three far-red fluorophores that can be used as robust mitochondrial photosensitizers to initiate mitophagy. The dyes offer maximal compatibility with multi-color live-cell imaging, as they do not spectrally overlap with commonly used fluorescent proteins. Through the use of these far-red fluorescent photosensitizers we found that mitophagic engulfment and mitophagosome maturation rates are highly correlated with the cellular Parkin-labeled mitochondria levels. This may represent a protective cellular mechanism to avoid membrane and lysosome depletion during mitophagy.

Highly sensitive cell imaging "Off-On" fluorescent probe for mitochondria and ATP.

A smart Off-On molecular scaffold/fluorescent probe 1 has been designed and synthesized. The probe has shown considerable photostability, cell permeability, organelle specificity and selectivity for ATP. The multicolor live cell imaging experiments in HeLa cells showed high selectivity of probe 1 for mitochondria with fluorescence "turn-on" response. As a proof of concept and promising prospects for application in biological sciences probe 1 has been utilized to detect ATP sensitively in a partial aqueous medium and intracellularly in HeLa cells. The favorable interaction between triphosphate unit of ATP and piperazine N atoms of probe 1 is attributed to synergistic effects of H-bonding and electrostatic interactions that encouraged the CH-π and π→π stacking between anthracene and purine rings. Consequently, the observed enhanced "turn-on" emission and a naked-eye sensitive blue-green color in the medium is attributable to arrest in photoinduced electron transfer (PET) process.

Internalization and Trafficking of Cell Surface Proteoglycans and Proteoglycan‐Binding Ligands

Using multicolor live cell imaging in combination with biochemical assays, we have investigated an endocytic pathway mediated by cell surface proteoglycans, primary receptors for many cationic ligands. We have characterized this pathway for a variety of proteoglycan-binding ligands including cationic polymers, lipids and polypeptides. Following clathrin- and caveolin-independent, but flotillin- and dynamin-dependent internalization, proteoglycan-bound ligands associate with flotillin-1-positive vesicles and are efficiently trafficked to late endosomes. The route to late endosomes differs considerably from that following clathrin-mediated endocytosis. The proteoglycan-dependent pathway to late endosomes does not require microtubule-dependent transport or phosphatidyl-inositol-3-OH kinase-dependent sorting from early endosomes. The pathway taken by these ligands is identical to that taken by an antibody against heparan sulfate proteoglycans, suggesting that this mechanism may be used generally by cell surface proteoglycans and proteoglycan-binding ligands that lack secondary receptors.

Fluorophores for live cell imaging of AGT fusion proteins across the visible spectrum

O6-alkylguanine-DNA alkyltransferase (AGT) fusion proteins can be specifically and covalently labeled with fluorescent O6-benzylguanine (O6-BG) derivatives for multicolor live cell imaging approaches. Here, we characterize several new BG fluorophores suitable for in vivo AGT labeling that display fluorescence emission maxima covering the visible spectrum from 472 to 673 nm, thereby extending the spectral limits set by fluorescent proteins. We show that the photostability of the cell-permeable dyes BG Rhodamine Green (BG505) and CP tetramethylrhodamine (CP-TMR) is in the range of enhanced green fluorescent protein (EGFP) and monomeric red fluorescent protein (mRFP), and that BG diethylaminomethyl coumarin (BGDEAC), a derivative of coumarin, is even more stable than enhanced cyan fluorescent protein (ECFP). Due to the increasing number of new BG derivatives with interesting fluorescence properties, such as far-red emission, fluorescence labeling of AGT fusion proteins is becoming a versatile alternative to existing live cell imaging approaches.