Bright Fluorophores Research Articles

Polymer dots enable deep in vivo multiphoton fluorescence imaging of microvasculature.

Deep in vivo imaging of vasculature requires small, bright, and photostable fluorophores suitable for multiphoton microscopy (MPM). Although semiconducting polymer dots (pdots) are an emerging class of highly fluorescent contrast agents with favorable advantages for the next generation of in vivo imaging, their use for deep MPM has never before been demonstrated. Herein, we characterize the multiphoton properties of three pdot variants and perform deep in vivo MPM imaging of cortical rodent microvasculature. We find pdot brightness exceeds conventional fluorophores, including quantum dots, and their broad multiphoton absorption spectrum permits imaging at wavelengths better-suited for biological imaging and confers compatibility with a range of longer excitation wavelengths. This results in substantial improvements in signal-to-background ratio (>3.5-fold) and greater cortical imaging depths (z = 1,300 µm). Ultimately, pdots are a versatile tool for MPM due to their extraordinary brightness and broad absorption, enabling interrogation of deep structures in vivo.

Conjugation of Fab' Fragments with Fluorescent Dyes for Single-Molecule Tracking On Live Cells.

Our understanding of the regulation and functions of cell-surface proteins has progressed rapidly with the advent of advanced optical imaging techniques. In particular, single-molecule tracking (SMT) using bright fluorophores conjugated to antibodies and wide-field microscopy methods such as total internal reflection fluorescence microscopy have become valuable tools to discern how endogenous proteins control cell biology. Yet, some technical challenges remain; in SMT, these revolve around the characteristics of the labeling reagent. A good reagent should have neutrality (in terms of not affecting the target protein's functions), tagging specificity, and a bright fluorescence signal. In addition, a long shelf-life is desirable due to the time and monetary costs associated with reagent preparation. Semiconductor-based quantum dots (Qdots) or Janelia Fluor (JF) dyes are bright and photostable, and are thus excellent candidates for SMT tagging. Neutral, high-affinity antibodies can selectively bind to target proteins. However, the bivalency of antibodies can cause simultaneous binding to two proteins, and this bridging effect can alter protein functions and behaviors. Bivalency can be avoided using monovalent Fab fragments generated by enzymatic digestion of neutral antibodies. However, conjugation of a Fab with a dye using the chemical cross-linking agent SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) requires reduction of the interchain disulfide bond within the Fab fragment, which can decrease the structural stability of the Fab and weaken its antigen-binding capability. To overcome this problem, we perform limited reduction of F(ab')2 to generate Fab' fragments using a weak reducer, cysteamine, which yields free sulfhydryl groups in the hinge region, while the interchain disulfide bond in Fab' is intact. Here, we describe a method that generates Fab' with high yield from two isoforms of IgG and conjugates the Fab' fragments with Qdots. This conjugation scheme can be applied easily to other types of dyes with similar chemical characteristics.

Generation of CoilR Probe Peptides for VIPER-labeling of Cellular Proteins.

Versatile Interacting Peptide (VIP) tags are a new class of genetically-encoded tag designed for imaging cellular proteins by fluorescence and electron microscopy. In 2018, we reported the VIPER tag ( Doh et al., 2018 ), which contains two elements: a genetically-encoded peptide tag (i.e., CoilE) and a probe peptide (i.e., CoilR). These two peptides deliver contrast to a protein of interest by forming a specific, high-affinity heterodimer. The probe peptide was designed with a single cysteine residue for site-specific modification via thiol-maleimide chemistry. This feature can be used to attach a variety of biophysical reporters to the peptide, including bright fluorophores for fluorescence microscopy or electron-dense nanoparticles for electron microscopy. In this Bio-Protocol, we describe our methods for expressing and purifying recombinant CoilR. Additionally, we describe protocols for making fluorescent or biotinylated probe peptides for labeling CoilE-tagged cellular proteins. This protocol is complemented by two other Bio-Protocols outlining the use of VIPER ( Doh et al., 2019a and 2019b).

Fast, in vivo voltage imaging using a red fluorescent indicator.

Genetically encoded voltage indicators (GEVIs) are emerging optical tools for acquiring brain-wide cell-type-specific functional data at unparalleled temporal resolution. To broaden the application of GEVIs in high-speed multispectral imaging, we used a high-throughput strategy to develop voltage-activated red neuronal activity monitor (VARNAM), a fusion of the fast Acetabularia opsin and the bright red fluorophore mRuby3. Imageable under the modest illumination intensities required by bright green probes (<50 mW mm-2), VARNAM is readily usable in vivo. VARNAM can be combined with blue-shifted optical tools to enable cell-type-specific all-optical electrophysiology and dual-color spike imaging in acute brain slices and live Drosophila. With enhanced sensitivity to subthreshold voltages, VARNAM resolves postsynaptic potentials in slices and cortical and hippocampal rhythms in freely behaving mice. Together, VARNAM lends a new hue to the optical toolbox, opening the door to high-speed in vivo multispectral functional imaging.

Developing a Bright NIR-II Fluorophore with Fast Renal Excretion and Its Application in Molecular Imaging of Immune Checkpoint PD-L1.

Fluorescence imaging in the second near-infrared (NIR-II) window holds impressive advantages of enhanced penetration depth and improved signal-to-noise ratio. Bright NIR-II fluorophores with renal excretion ability and low tissue accumulation are favorable for in vivo molecular imaging applications as they can render the target-mediated molecular imaging process easily distinguishable. Here, a probe (anti-PD-L1-BGP6) comprising a fluorophore (IR-BGP6) covalently bonded to the programmed cell death ligand-1 monoclonal antibody (PD-L1 mAb) for molecular imaging of immune checkpoint PD-L1 (a targeting site upregulated in various tumors for cancer imaging) in the NIR-II window is reported. Through molecular optimization, the bright NIR-II fluorophore IR-BGP6 with fast renal excretion (≈91% excretion in general through urine within the first 10 h postinjection) is developed. The conjugate anti-PD-L1-BGP6 succeeds in profiling PD-L1 expression and realizes efficient noninvasive molecular imaging in vivo, achieving a tumor to normal tissue (T/NT) signal ratio as high as ≈9.5. Compared with the NIR-II fluorophore with high nonspecific tissue accumulation, IR-BGP6 derived PD-L1 imaging significantly enhances the molecular imaging performance, serving as a strong tool for potentially studying underlying mechanism of immunotherapy. The work provides rationales to design renal-excreted NIR-II fluorophores and illustrate their advantages for in vivo molecular imaging.

A Highly Photostable Near-Infrared Labeling Agent Based on a Phospha-rhodamine for Long-Term and Deep Imaging.

Various fluorescence microscopy techniques require bright NIR-emitting fluorophores with high chemical and photostability. Now, the significant performance improvement of phosphorus-substituted rhodamine dyes (PORs) upon substitution at the 9-position with a 2,6-dimethoxyphenyl group is reported. The thus obtained dye PREX 710 was used to stain mitochondria in living cells, which allowed long-term and three-color imaging in the vis-NIR range. Moreover, the high fluorescence longevity of PREX 710 allows tracking a dye-labeled biomolecule by single-molecule microscopy under physiological conditions. Deep imaging of blood vessels in mice brain has also been achieved using the bright NIR-emitting PREX 710-dextran conjugate.

Homogeneous Time-resolved Förster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion.

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous insulin assays have existed for decades, the recent advent of homogeneous time-resolved Förster Resonance Energy Transfer (HTRF) technology has significantly simplified these measurements. This is a rapid, cost-effective, reproducible, and robust optical assay reliant upon antibodies conjugated to bright fluorophores with long lasting emission which facilitates time-resolved Förster Resonance Energy Transfer. Moreover, HTRF insulin detection is amenable for the development of high-throughput screening assays. Here we use HTRF to detect insulin secretion in INS-1E cells, a rat insulinoma-derived cell line. This allows us to estimate basal levels of insulin and their changes in response to glucose stimulation. In addition, we use this insulin detection system to confirm the role of dopamine as a negative regulator of glucose-stimulated insulin secretion (GSIS). In a similar manner, other dopamine D2-like receptor agonists, quinpirole, and bromocriptine, reduce GSIS in a concentration-dependent manner. Our results highlight the utility of the HTRF insulin assay format in determining the role of numerous drugs in GSIS and their pharmacological profiles.

Synthesis, Crystal Structure, and the Deep Near-Infrared Absorption/Emission of Bright AzaBODIPY-Based Organic Fluorophores.

Annularly fused azaBODIPY-based organic fluorophores (HBPs 2) containing up to 13 aromatic ring fusions were synthesized by a Suzuki coupling reaction with bromoazadipyrromethenes and a subsequent regioselective oxidative ring-fusion reaction. X-ray analysis indicates almost planar dipyrrin cores for all crystals but overall curved or "wave" conformations for those HBP dyes. These molecules exhibit unique structural and physical properties including excellent spectral selectivity (negligible absorption between 300 and 700 nm), sharp near-infrared (NIR) absorption (up to 878 nm) and emission (up to 907 nm), large extinction coefficient (up to 4.5 × 105 M-1 cm-1), and excellent photostabilities.

Super-Chelators for Advanced Protein Labeling in Living Cells.

Live-cell labeling, super-resolution microscopy, single-molecule applications, protein localization, or chemically induced assembly are emerging approaches, which require specific and very small interaction pairs. The minimal disturbance of protein function is essential to derive unbiased insights into cellular processes. Herein, we define a new class of hexavalent N-nitrilotriacetic acid (hexaNTA) chelators, displaying the highest affinity and stability of all NTA-based small interaction pairs described so far. Coupled to bright organic fluorophores with fine-tuned photophysical properties, the super-chelator probes were delivered into human cells by chemically gated nanopores. These super-chelators permit kinetic profiling, multiplexed labeling of His6 - and His12 -tagged proteins as well as single-molecule-based super-resolution imaging.

Super‐Chelators for Advanced Protein Labeling in Living Cells

AbstractLive‐cell labeling, super‐resolution microscopy, single‐molecule applications, protein localization, or chemically induced assembly are emerging approaches, which require specific and very small interaction pairs. The minimal disturbance of protein function is essential to derive unbiased insights into cellular processes. Herein, we define a new class of hexavalent N‐nitrilotriacetic acid (hexaNTA) chelators, displaying the highest affinity and stability of all NTA‐based small interaction pairs described so far. Coupled to bright organic fluorophores with fine‐tuned photophysical properties, the super‐chelator probes were delivered into human cells by chemically gated nanopores. These super‐chelators permit kinetic profiling, multiplexed labeling of His6‐ and His12‐tagged proteins as well as single‐molecule‐based super‐resolution imaging.

Redesigning the Coumarin Scaffold into Small Bright Fluorophores with Far-Red to Near-Infrared Emission and Large Stokes Shifts Useful for Cell Imaging.

Among the palette of previously described fluorescent organic molecules, coumarins are ideal candidates for developing cellular and molecular imaging tools due to their high cell permeability and minimal perturbation of living systems. However, blue-to-cyan fluorescence emission is usually difficult in in vivo applications due to the inherent toxicity and poor tissue penetration of short visible light wavelengths. Here, we introduce a new family of coumarin-based fluorophores, nicknamed COUPY, with promising photophysical properties, including emission in the far-red/near-infrared (NIR) region, large Stokes shifts, high photostability, and excellent brightness. COUPY fluorophores were efficiently synthesized in only three linear synthetic steps from commercially available precursors, with the N-alkylation of a pyridine moiety being the key step at the end of the synthetic route, as it allows for the tuning of the photophysical properties of the resulting dye. Owing to their low molecular weights, COUPY dyes show excellent cell permeability and accumulate selectively in nucleoli and/or mitochondria of HeLa cells, as their far-red/NIR fluorescence emission is easily detected at a concentration as low as 0.5 μM after an incubation of only 20 min. We anticipate that these coumarin scaffolds will open a way to the development of novel coumarin-based far-red to NIR emitting fluorophores with potential applications for organelle imaging and biomolecule labeling.

Linear dendronized polyols as a multifunctional platform for a versatile and efficient fluorophore design

Linear dendronized polyols (LDPs)as a modular platform for bright, stable, and biocompatible polymeric fluorophores applicable for fluorescent bioimaging studies.

Spatiotemporal Fluctuation Analysis of Molecular Diffusion Laws in Live-Cell Membranes.

A present challenge of membrane biophysics is deciphering the dynamic behavior of molecules, such as lipids and proteins, within the natural environment of a living-cell membrane. Here, a fluorescence fluctuation-based approach will be described, which makes it possible to probe the "diffusion law" of molecules directly from imaging, in the form of a mean square displacement vs time-delay plot (iMSD), with no need for interpretative models. Of note, the presented approach does not require extraction of the molecular trajectories nor the use of bright fluorophores. Conversely, it can be used at high fluorophore density and with relatively dim fluorophores, such as GFP-tagged molecules transiently expressed within cells. The ability of this approach to resolve average molecular dynamic properties well below the diffraction limit will be discussed. Overall, this novel approach is proposed as a powerful tool for the determination of kinetic and thermodynamic parameters over wide spatial and temporal scales.

A]-Phenanthrene-Fused BF2 Azadipyrromethene (AzaBODIPY) Dyes as Bright Near-Infrared Fluorophores.

A new substitution pattern of BF2 azadipyrromethene (azaBODIPY) dyes was obtained by phenanthrene fusion through a key palladium-catalyzed intramolecular C-H activation reaction. These [a]-phenanthrene-fused azaBODIPYs have a near planar structure of the phenanthrene-fused azadipyrromethene core in the crystalline state. The chromophore absorbs (log ε > 5) and fluoresces (ϕ = 0.32-0.38) strongly above 700 nm with excellent photostability and may be used as an attractive bright NIR bioimaging agent.

Flavylium Polymethine Fluorophores for Near- and Shortwave Infrared Imaging.

Bright fluorophores in the near-infrared and shortwave infrared (SWIR) regions of the electromagnetic spectrum are essential for optical imaging in vivo. In this work, we utilized a 7-dimethylamino flavylium heterocycle to construct a panel of novel red-shifted polymethine dyes, with emission wavelengths from 680 to 1045 nm. Photophysical characterization revealed that the 1- and 3-methine dyes display enhanced photostability and the 5- and 7-methine dyes exhibit exceptional brightness for their respective spectral regions. A micelle formulation of the 7-methine facilitated SWIR imaging in mice. This report presents the first polymethine dye designed and synthesized for SWIR in vivo imaging.

Abstract 4991: Dynamic reprogramming of the chromatin landscape in cancer: Studies in real time

Abstract Reprogramming of the chromatin landscape by sequence-specific transcription factors (TF) can influence in the prevalence and progression of several diseases, including cancers. Steroid receptors are a class of hormone-regulated TF that have a major impact on the progression of cancers. The link between the androgen receptor (AR) action in prostate cancer and the glucocorticoid (GR) and estrogen receptor (ER) action in breast cancer is well-known. Furthermore, other TFs such as the pioneer factor FoxA1 has been extensively linked to AR and ER action in these cancers. Although the action of these TF have been widely studied, the knowledge is almost exclusively based on population-based assays such as ChIP-seq and DHS-seq. Hence, the dynamic binding behavior of these factors in living cell remains unclear. For example, the classical pioneer factor model suggests long term static binding events between pioneer TFs, such as FoxA1, and chromatin. However, little is known on the dynamic action of FoxA1 in living cells. We have examined the behavior of steroid receptors and cofactors, including FoxA1, at the single-molecule level in living cells. These factors were fused with HaloTag, and labeled with the bright and stable fluorophore JF549. Single-molecule tracking (SMT) of these proteins (using HILO microscopy) revealed a highly dynamic binding behavior of TFs. This supports transient rather than stable TF chromatin interactions. Specifically, two distinct binding populations have been observed for all factors tested; fast or slow stops. Fast stops represent genomic scanning of TF, while slow stops, with residence time of 6-14 sec, represent functional binding events at specific response elements. The vast majority of molecules at any given time are either diffusing or exhibit fast stops, while only a small percentage exhibit slow stops. However, hormone activation of AR, GR or ER results in an increase in the percentage of molecules with slow stops compared to the unstimulated state. Thus, our results indicate that on a single-molecule level, only a small proportion TFs are functionally bound at any given time. In addition to affirming the general model that many TFs are highly dynamic in their chromatin binding activity, SMT experiments in live cells revealed a highly dynamic interaction of FoxA1 with chromatin in vivo. Unexpectedly, we also observed by ChIP-seq and DHS-seq analysis that at subset of genomic sites, the role of pioneer can be reversed, with the steroid receptors serving to enhance binding of FoxA1. We propose a general model, dynamic assisted loading, wherein interactions between TFs and pioneer factors are highly dynamic. Our findings show that the action of key TFs in cancer development are more complex and dynamic in real time than previously reported. Citation Format: Ville Paakinaho, Diego M. Presman, Erin E. Swinstead, Tina B. Miranda, David A. Ball, Tatiana S. Karpova, Gordon L. Hager. Dynamic reprogramming of the chromatin landscape in cancer: Studies in real time [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4991. doi:10.1158/1538-7445.AM2017-4991

Synthesis and crystal structure of a meso-decene-BODIPY dye as a functional bright fluorophore for silicone matrices

A meso-decene-BODIPY dye was synthesized and conjugated to a polydimethylsiloxane. Fluorescence properties of the obtained polymer drastically changed in different solvents. The polymer showed an unusual for other siloxane polymers trend to structuration.

T Lymphocyte Immunophenotyping: 14-Color Flow Cytometry Panel Design Using the Attune NxT Flow Cytometer and Super Bright Fluorescent Dyes

Abstract The Invitrogen™ eBioscience™ Super Bright polymer dyes represent a suite of bright fluorophores excited by the violet laser (405 nm). Optimized for use in flow cytometry, the Super Bright dyes allow for expanded use of violet laser excitation and promote streamlined multicolor panel design. Due to their inherent brightness, detection of cell populations with low abundance targets is possible. A 14-color panel used to characterize human T-cell lymphocytes is presented, with information on differentiation profiles, activation or exhaustion status, and co-stimulation activity. Both cryopreserved human peripheral blood mononuclear cells (PBMC) and fresh blood collected from a normal donor was used. Acquisition and analysis was performed using the Attune™ NxT Flow Cytometer four laser system, which has the ability to detect multiple violet-excited fluorophores. The results show well characterized cell populations, with expected changes between unstimulated and stimulated PBMC ex vivo. Correction for spectral overlap was performed using standard instrument auto-compensation procedures. The unique acoustic focusing technology of Attune NxT Flow Cytometer enables rapid and accurate detection with sensitivity at high flow rates without compromise of data quality, as demonstrated with this 14-color data set. The Attune NxT cytometer is designed to accommodate a wide variety of experimental conditions and combined with the Super Bright dyes provides expanded choice for panel design. For research use only. Not for use in diagnostic procedures.

Bright, Stable, and Biocompatible Organic Fluorophores Absorbing/Emitting in the Deep Near‐Infrared Spectral Region

AbstractSmall‐molecule organic fluorophores spectrally active in the 800–950 nm region are sought‐after for their broad potential in biomedical and material applications. We have developed a new family of brightly fluorescent dyes (ECX) to meet this challenge. ECX dyes are transparent to the visible region, while strongly absorbing in the NIR region at approximately 880 nm. They emit at around 915 nm with a fluorescence quantum yield up to 13.3 %. ECX dyes exhibit high chemostability, high photostability, and low tendency to aggregate. Other merits of ECX dyes include low degree of solvatochromism and facile post‐synthetic derivatization. ECX dyes potentially make available the 800–950 nm region for spectroscopic and microscopic applications and are also expected to find broad material applications.

Bright and Photostable Fluorophores for Advanced Fluorescence Microscopy

Advanced fluorescence microscopy, including single-molecule and super-resolution imaging, demands bright and photostable fluorophores. We have recently reported a general approach to improve fluorophores brightness in living cells by substituting the N,N-dimethylamino groups found in classic dyes with four-membered azetidine rings (Nature Methods 12, 244250 (2015)). In an unpublished work we have synthesized new derivatives containing substituents on the azetidine ring. Using this approach we were able to fine tune the wavelength and fluorogenecity of the fluorophore without affecting brightness. Here, we report that several of these novel substituted-azetidine fluorophores, as well as the substituted-xanthene ones, exhibit substantial improvements in photostability in living cells. These fluorophores enable robust multi-color, wash-free imaging with a large photon budget. We are investigating their phototoxicity and mechanism in order to maximize the photostability, to generalize this approach to different fluorophores, and to apply these fluorophores to diverse biological settings, including living cells, tissues, and animals. We freely share our fluorophores with the academic community. This work is supported by HHMI and NIH (U01 EB 021236).