Dye Alexa Fluor Research Articles

Multicolor, Cell-Impermeable, and High Affinity BACE1 Inhibitor Probes Enable Superior Endogenous Staining and Imaging of Single Molecules.

The prevailing but not undisputed amyloid cascade hypothesis places the β-site of APP cleaving enzyme 1 (BACE1) center stage in Alzheimer's Disease pathogenesis. Here, we investigated functional properties of BACE1 with novel tag- and antibody-free labeling tools, which are conjugates of the BACE1-inhibitor IV (also referred to as C3) linked to different impermeable Alexa Fluor dyes. We show that these fluorescent small molecules bind specifically to BACE1, with a 1:1 labeling stoichiometry at their orthosteric site. This is a crucial property especially for single-molecule and super-resolution microscopy approaches, allowing characterization of the dyes' labeling capabilities in overexpressing cell systems and in native neuronal tissue. With multiple colors at hand, we evaluated BACE1-multimerization by Förster resonance energy transfer (FRET) acceptor-photobleaching and single-particle imaging of native BACE1. In summary, our novel fluorescent inhibitors, termed Alexa-C3, offer unprecedented insights into protein-protein interactions and diffusion behavior of BACE1 down to the single molecule level.

Abstract 3108: Antibody labeling reagents to rapidly screen for the binding and internalization of therapeutic antibodies

Abstract Introduction: There is a growing need in the immunotherapy research field for tools to rapidly screen novel therapeutic antibodies, whether looking for cells expressing IgG, target binding, or tracking internalization. Thermo Fisher Scientific has developed the next generation of Invitrogen™ Zenon™ reagents to enable screening of potential therapeutic antibodies, working across a variety of sample types including primary B cells and hybridomas, while being compatible with a wide range of platforms including flow cytometry, high content imaging, and in-incubator imaging systems. Methods: The Invitrogen™ Zenon™ Alexa Fluor™ Plus IgG Labeling Reagents to screen for antibody binding provide a fast, versatile, and reliable method to evaluate antibody binding to cell surface antigens. Zenon screening reagents are labeled with Invitrogen Alexa Fluor Plus 488, Alexa Fluor Plus 594, or Alexa Fluor Plus 647 dyes (i.e., labeling reagent). The labeling reagent is directed against the Fc portion of an intact mouse or human IgG primary antibody. The formation of the zenon-antibody complex is rapid and requires no purification steps. The Invitrogen™ Zenon™ pHrodo™ Deep Red Labeling Reagents for screening antibody internalization provides a highly sensitive method for monitoring antibody internalization. Zenon pHrodo Deep Red utilizes the same screening reagent as Zenon Alexa Fluor Plus reagents, replacing Alexa fluor dyes with the pH-sensitive dye, pHrodo Deep Red. The pHrodo Deep Red sensors have minimal fluorescence at neutral pH and only upon entry into the late endosome and lysosome do they brightly fluoresce. Zenon pHrodo Deep Red Labeling Reagents are perfectly suited for screening the endocytosis and degradation of potentially therapeutic antibodies including antibody-drug conjugates. Results: Live cells stained with Zenon-labeled antibodies show remarkable signal to background in both flow and imaging applications, while retaining their signal following fixation. Therapeutic antibodies such as anti-Her2 labeled with Zenon pHrodo Deep Red show as bright fluorescent puncta upon lysosomal degradation in the Alexa Fluor 647 channel and co-localize with lysosomal markers such as Invitrogen™ LysoTracker Red dye. Both Zenon Alexa Fluor Plus and Zenon phrodo Deep Red are ideal reagents for screening antibody panels of up to 96 different antibodies. Conclusions: Thermo Fisher Scientific has developed sensitive and rapid Zenon reagents to allow for high throughput therapeutic antibody screening. In addition to Zenon reagents, Thermo Fisher Scientific has developed additional tools that allow for specific monitoring of antibody and protein uptake into the lysosome for degradation. These tools not only greatly improve the workflow from our existing portfolio, but allow for increased sensitivity allowing the field to make decisive decisions when selecting candidate immunotherapies. Acknowledgments: I would like to thank the whole team that worked to develop these new labeling reagents including: Maria Olson and Chris Langsdorf. Citation Format: Ryan W. Holly. Antibody labeling reagents to rapidly screen for the binding and internalization of therapeutic antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3108.

A multiplex immunoprofiling approach for detecting the co-localization of breast cancer biomarkers using a combination of Alexafluor - Quantum dot conjugates and a panel of chromogenic dyes

There is a plethora of information embedded in a tissue section that the conventional IHC understands only partially. Predictive biomarkers for precision immuno-oncology heavily dependent on the spatial arrangement of cells and the co-expression patterns in the tissue sections. Here we have explored the versatility of indirect multiplex immunofluorescence (mIF) and indirect multiplex immunohistochemistry (mIHC) for the labeling of breast cancer prognostic markers in routinely processed, formalin-fixed paraffin-embedded (FFPE) tissues at high resolution. The multiplex immunohistochemistry protocol utilized sequential staining for the chromogenic immunolabelling of Estrogen Receptor α (ERα) or Progesterone Receptor (PR), Human Epidermal Growth Factor Receptor 2 (HER2), and Nucleoside diphosphate kinase 1 (NM23) by multicolor chromogens in different combinations. A feasible workflow for multiplex immunofluorescence was also effectively standardized for ERα, PR, and HER2 using combinations of commercially available Alexa Fluor and Quantum dots semiconductor nanocrystal conjugated secondary antibodies. Multiplex chromogenic immunolabeling revealed differential expression of the markers on the same slide. Kappa statistics revealed perfect agreement with uniplex immunohistochemistry. For multiplex fluorescence approach, surface receptor detection using Quantum dots and Alexa fluor dyes for cytoplasmic or nuclear markers performed well for profiling multiple co-localized biomarkers on a single paraffin tissue section. The technique developed reveals additional information such as co-expression, spatial relationships, and tumor heterogeneity, providing a deeper insight into developing combinatorial therapeutic strategies in clinical care. This high throughput workflow complements the outcomes of traditional IHC while saving tissue, time, labour, and reagents.

Synthesis of Short Peptides with Perfluoroalkyl Side Chains and Evaluation of Their Cellular Uptake Efficiency.

With an increasing demand for macromolecular biotherapeutics, the issue of their poor cell-penetrating abilities requires viable and relevant solutions. Herein, we report tripeptides bearing an amino acid with a perfluoroalkyl (RF ) group adjacent to the α-carbon. RF -containing tripeptides were synthesized and evaluated for their ability to transport a conjugated hydrophilic dye (Alexa Fluor 647) into the cells. RF -containing tripeptides with the fluorophore showed high cellular uptake efficiency and none of them were cytotoxic. Interestingly, we demonstrated that the absolute configuration of perfluoroalkylated amino acids (RF -AAs) affects not only nanoparticle formation but also the cell permeability of the tripeptides. These novel RF -containing tripeptides are potentially useful as short and noncationic cell-penetrating peptides (CPPs).

Click-Functionalization of Silanized Carbon Nanotubes: From Inorganic Heterostructures to Biosensing Nanohybrids.

Here we present an approach to functionalize silanized single-walled carbon nanotubes (SWNTs) through copper-free click chemistry for the assembly of inorganic and biological nanohybrids. The nanotube functionalization route involves silanization and strain-promoted azide-alkyne cycloaddition reactions (SPACC). This was characterized by X-ray photoelectron spectroscopy, scanning electron microscopy, transmission electron microscopy, Raman spectroscopy and Fourier transform infra-red spectroscopy. Silane-azide-functionalized SWNTs were immobilized from solution onto patterned substrates through dielectrophoresis (DEP). We demonstrate the general applicability of our strategy for the functionalization of SWNTs with metal nanoparticles (gold nanoparticles), fluorescent dyes (Alexa Fluor 647) and biomolecules (aptamers). In this regard, dopamine-binding aptamers were conjugated to the functionalized SWNTs to perform real-time detection of dopamine at different concentrations. Additionally, the chemical route is shown to selectively functionalize individual nanotubes grown on the surface of silicon substrates, contributing towards future nano electronic device applications.

CD38-specific nanobodies allow in vivo imaging of multiple myeloma under daratumumab therapy.

Recent studies have demonstrated the feasibility of CD38-specific antibody constructs for in vivo imaging of multiple myeloma. However, detecting multiple myeloma in daratumumab-pretreated patients remains difficult due to overlapping binding epitopes of the CD38-specific imaging antibody constructs and daratumumab. Therefore, the development of an alternative antibody construct targeting an epitope of CD38 distinct from that of daratumumab is needed. We report the generation of a fluorochrome-conjugated nanobody recognizing such an epitope of CD38 to detect myeloma cells under daratumumab therapy in vitro, ex vivo, and in vivo. We conjugated the CD38-specific nanobody JK36 to the near-infrared fluorescent dye Alexa Fluor 680. The capacity of JK36AF680 to bind and detect CD38-expressing cells pretreated with daratumumab was evaluated on CD38-expressing tumor cell lines in vitro, on primary myeloma cells from human bone marrow biopsies ex vivo, and in a mouse tumor model in vivo. Fluorochrome-labeled nanobody JK36AF680 showed specific binding to CD38-expressing myeloma cells pretreated with daratumumab in vitro and ex vivo and allowed for specific imaging of CD38-expressing xenografts in daratumumab-pretreated mice in vivo. Our study demonstrates that a nanobody recognizing a distinct, non-overlapping epitope of CD38 allows the specific detection of myeloma cells under daratumumab therapy in vitro, ex vivo, and in vivo.

Fluorescent A2A and A3 adenosine receptor antagonists as flow cytometry probes.

Adenosine receptor (AR) ligands are beingdeveloped for metabolic, cardiovascular, neurological, and inflammatory diseases and cancer. The ease of drug discovery is contingent on the availability of pharmacological tools. Fluorescent antagonist ligands for the human A2A and A3ARs were synthesized using two validated pharmacophores, 1,3-dipropyl-8-phenylxanthine and triazolo[1,5-c]quinazolin-5-yl)amine, which were coupled toeight reporter fluorophores: AlexaFluor, JaneliaFluor (JF), cyanine, and near infrared (NIR) dyes. The conjugates were first screened using radioligand binding in HEK293 cells expressing one of the three AR subtypes. The highest affinities at A2AAR were Ki 144-316nM for 10, 12, and 19, and at A3AR affinity of Ki 21.6nM for 19. Specific binding of JF646 conjugate MRS7774 12 to the HEK293 cell surface A2AAR was imaged using confocal microscopy. Compound 19MRS7535, a triazolo[1,5-c]quinazolin-5-yl)amine containing a Sulfo-Cy7 NIR dye, was suitable for A3AR characterization in whole cells by flow cytometry (Kd 11.8nM), and its bitopic interaction mode with an A3AR homology model was predicted. Given its affinity and selectivity (11-fold vs. A2AAR, ~ 50-fold vs. A1AR and A2BAR) and a good specific-to-nonspecific binding ratio, 19 could be useful for live cell or potentially a diagnostic in vivo NIR imaging tool and/or therapy targeting the A3AR.

Total Internal Reflection of Two Lasers in a Single Planar Optical Waveguide

Multiplex biomarker quantitation is ideal for tracking the progression of a disease, but sensitive, specific, and quantitative multiplex biosensing remains challenging. We achieved total internal reflection with two lasers in a single planar optical waveguide by coupling 532 nm laser light into the diffraction grating of a waveguide and 635 nm laser light directly into the thin film of the same waveguide. We confirmed that the evanescent fields generated in the waveguide can excite two fluorescent dyes (Alexa Fluor 532 and Alexa Fluor 647) held to the surface of the waveguide through biotin-streptavidin chemistry. This sensing concept holds significant potential as a platform for multiplex biomarker detection.

Organotropic dendrons with high potency as HIV-1, HIV-2 and EV-A71 cell entry inhibitors

We have recently described a novel family of compounds of reduced size and dual anti-HIV and anti-EV71 activity that encompasses tripodal and tetrapodal derivatives. The tripodal prototype, AL-470, has a nitro group at the focal point of the central scaffold and three attached tryptophan residues, each of which bearing an isophthaloyl moiety at the C2 position of the indole ring. A nitro to amino substitution has allowed us now to introduce a chemically addressable functionality to perform further structural modifications consisting of both direct and linker-mediated attachment of several aromatic groups, including the fluorescent dye Alexa Fluor 647 and the antibody-recruiting 2,4-dinitrophenyl motif. Some of the derivatives turned out to be more potent and selective than AL-470 against HIV-1, HIV-2 and EV-A71. The fluorescent probe demonstrated a specific tropism for intestines and lungs, two important niches for the human microbiome in health and disease.

PH and ionic strength triggered destabilization of biocompatible stable water-in-oil-in-water (W/O/W) emulsions

The design of biocompatible multiple emulsions is an important challenge in the field of controlled delivery systems for protecting and delivering compounds encapsulated and protected in the innermost phase. In this paper, we use biocompatible water – Miglyol®812 water-in-oil-in-water (W/O/W) emulsions stabilized by a stimuli-responsive diblock copolymer consisting of poly(dimethylsiloxane) (PDMS) and poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) to design an easy-to-process new delivery W/O/W system. Such emulsions are formed in a single emulsification step. They present a high encapsulation yield and are shown to be stable over months. As such, the encapsulation of a hydrophilic dye (Alexa fluor) in the innermost water phase is successfully demonstrated over months. These emulsions are stimulable either by a shift in pH level or in ionic strength. The former destabilizes the multiple emulsion and leads to a simple one while the latter partly maintains the multiple character. Eventually both stimulations are effective in the dye release and molecular mechanisms are proposed for explaining the observed two-stage kinetics of release. Triggered release of a hydrophilic dye encapsulated in a biocompatible W/O/W emulsion, formed by one-step mixing and stabilized by a single block copolymer using pH and ionic strength ( I ) stimuli. Oil phases appear in blue, dye-loaded aqueous phases appear in red and dye-free aqueous phases appear in black.

Glucose-responsive oral insulin delivery platform for one treatment a day in diabetes

Glucose-responsive oral insulin delivery platform for one treatment a day in diabetes

Two-photon peak molecular brightness spectra reveal long-wavelength enhancements of multiplexed imaging depth and photostability.

The broad use of two-photon microscopy has been enabled in part by Ti:Sapphire femtosecond lasers, which offer a wavelength-tunable source of pulsed excitation. Action spectra have thus been primarily reported for the tunable range of Ti:Sapphire lasers (∼700-1000 nm). However, longer wavelengths offer deeper imaging in tissue via reduced scattering and spectral dips in water absorption, and new generations of pulsed lasers offer wider tunable ranges. We present the peak molecular brightness spectra for eight Alexa Fluor dyes between 700-1300 nm as a first-order surrogate for action spectra measured with an unmodified commercial microscope, which reveal overlapping long-wavelength excitation peaks with potential for multiplexed excitation. We demonstrate simultaneous single-wavelength excitation of six spectrally overlapping fluorophores using either short (∼790 nm) or long (∼1090 nm) wavelengths, and that the newly characterized excitation peaks measured past 1000 nm offer improved photostability and enhanced fidelity of linear spectral unmixing at depth compared to shorter wavelengths.

Molecular and Spectroscopic Characterization of Green and Red Cyanine Fluorophores from the Alexa Fluor and AF Series.

The front cover artwork was provided by Gabriel Moya, a PhD student in the Cordes lab (LMU Biocenter Munich, Germany). The cover image shows an artistic impression of the chemical structures of two fluorophores investigated in the paper. Read the full text of the Article at 10.1002/cphc.202000935.

Investigating Single-Molecule Fluorescence Spectral Heterogeneity of Rhodamines Using High-Throughput Single-Molecule Spectroscopy.

We experimentally investigated several intramolecular coordinate and environmental changes as potential causes of single-molecule fluorescence spectral heterogeneities (smFSH). We developed a high-throughput single-molecule spectroscopy method to analyze more than 5000 single-molecule emission spectra from each of 9 commonly used fluorophores with different structural rigidities and deposited on substrates with different polarities. We observed an unexpectedly high smFSH from structurally rigid Rhodamine B compared with a structurally flexible Cyanine dye-Alexa Fluor 647. Based on experimentally measured smFSH, we ruled out the system's noise uncertainty, single-molecule spectral diffusion, and environmental polarity as the primary causes of the high smFSH. We found that the rotational flexibility of N,N-dialkylated groups contributed to the smFSH. With the high smFSH observed in structurally more rigid model fluorophores, we speculated that other intramolecular coordinate and environmental changes might also contribute to the high smFSH in Rhodamines.

Switching behaviour of dSTORM dyes in glycerol-containing buffer

To suppress optical aberrations caused by refractive index mismatch, we employ glycerol-immersion objectives in conjunction with fused silica cover glasses and imaging buffers with a high glycerol content. Here we demonstrate that the addition of glycerol to the buffer does not degrade the switching behaviour of the dyes Alexa Fluor 647 and Alexa Fluor 568 in dSTORM measurements, which shows that this approach is suitable for dSTORM. Additionally, we report evidence that sealed sample geometries as used in our experiments reduce photobleaching due to the lower influx of oxygen into the imaging buffer.

Multidonor surface energy transfer from Alexa Fluor dyes to gold nanoparticles: a quest for innovative sensor applications

We report energy transfer (ET) from two dyes: Alexa Fluor 514 (AF514) and Alexa Fluor 532 (AF532) to gold nanoparticles (AuNPs) of three different sizes (10, 30, and 53 nm) employing steady-state and time-resolved fluorescence measurements. The results show that the fluorescence intensity and fluorescence lifetimes of donor (D) molecules AF514 and AF532 decrease with increase in the concentration of acceptor (A) AuNPs (2 to 10 μM) upon interaction with AuNPs, thereby confirming the occurrence of ET between D and A. This clearly suggests that these two Alexa Fluor molecules act as efficient donors and AuNPs as excellent acceptors. Interestingly, the Forster distance (Ro) determined for these dyes varies from 212 to 550 Å with increasing size of AuNPs and suggests that the ET from AF514 and AF532 to AuNPs is essentially obeying surface energy transfer (SET) process following 1 / d4 distance dependence. As is well known, Forster resonance energy transfer is efficient for separation distances of up to 100 Å, beyond which its efficiency decreases. Thus, the present results follow dipole-surface type ET from molecule dipole (AF514 and AF532) to nanometal (Au) surface. The influence of size and distance on the SET from AF514 and AF532 to AuNPs is discussed. Further, the quenching of donor fluorescence in the presence of AuNPs and nonradiative ET are analyzed using Stern–Volmer plots. Our study is an experimental quest to explore the potential of such dye–noble metal NPs pairs performing as sensitive chemical and biosensors.

CD24-targeted intraoperative fluorescence image-guided surgery leads to improved cytoreduction of ovarian cancer in a preclinical orthotopic surgical model.

BackgroundThe completeness of resection is a key prognostic indicator in patients with ovarian cancer, and the application of tumour-targeted fluorescence image-guided surgery (FIGS) has led to improved detection of peritoneal metastases during cytoreductive surgery. CD24 is highly expressed in ovarian cancer and has been shown to be a suitable biomarker for tumour-targeted imaging.MethodsCD24 expression was investigated in cell lines and heterogenous patient-derived xenograft (PDX) tumour samples of high-grade serous ovarian carcinoma (HGSOC). After conjugation of the monoclonal antibody CD24 to the NIR dye Alexa Fluor 750 and the evaluation of the optimal pharmacological parameters (OV-90, n = 21), orthotopic HGSOC metastatic xenografts (OV-90, n = 16) underwent cytoreductive surgery with real-time feedback. The impact of intraoperative CD24-targeted fluorescence guidance was compared to white light and palpation alone, and the recurrence of disease was monitored post-operatively (OV-90, n = 12). CD24-AF750 was further evaluated in four clinically annotated orthotopic PDX models of metastatic HGSOC, to validate the translational potential for intraoperative guidance.FindingsCD24-targeted intraoperative NIR FIGS significantly (47•3%) improved tumour detection and resection, and reduced the post-operative tumour burden compared to standard white-light surgery in orthotopic HGSOC xenografts. CD24-AF750 allowed identification of minuscule tumour lesions which were undetectable with the naked eye in four HGSOC PDX.InterpretationCD24-targeted FIGS has translational potential as an aid to improve debulking surgery of ovarian cancer.FundingThis study was supported by the H2020 program MSCA-ITN [675743], Helse Vest RHF, and Helse Bergen HF [911809, 911852, 912171, 240222, 911974, HV1269], as well as by The Norwegian Cancer Society [182735], and The Research Council of Norway through its Centres of excellence funding scheme [223250, 262652].

Surface passivation of zero-mode waveguide nanostructures: benchmarking protocols and fluorescent labels

Zero mode waveguide (ZMW) nanoapertures efficiently confine the light down to the nanometer scale and overcome the diffraction limit in single molecule fluorescence analysis. However, unwanted adhesion of the fluorescent molecules on the ZMW surface can severely hamper the experiments. Therefore a proper surface passivation is required for ZMWs, but information is currently lacking on both the nature of the adhesion phenomenon and the optimization of the different passivation protocols. Here we monitor the influence of the fluorescent dye (Alexa Fluor 546 and 647, Atto 550 and 647N) on the non-specific adhesion of double stranded DNA molecule. We show that the nonspecific adhesion of DNA double strands onto the ZMW surface is directly mediated by the organic fluorescent dye being used, as Atto 550 and Atto 647N show a pronounced tendency to adhere to the ZMW while the Alexa Fluor 546 and 647 are remarkably free of this effect. Despite the small size of the fluorescent label, the surface charge and hydrophobicity of the dye appear to play a key role in promoting the DNA affinity for the ZMW surface. Next, different surface passivation methods (bovine serum albumin BSA, polyethylene glycol PEG, polyvinylphosphonic acid PVPA) are quantitatively benchmarked by fluorescence correlation spectroscopy to determine the most efficient approaches to prevent the adsorption of Atto 647N labeled DNA. Protocols using PVPA and PEG-silane of 1000 Da molar mass are found to drastically avoid the non-specific adsorption into ZMWs. Optimizing both the choice of the fluorescent dye and the surface passivation protocol are highly significant to expand the use of ZMWs for single molecule fluorescence applications.

Ultrastructure of light-activated axons following optogenetic stimulation to produce late-phase long-term potentiation.

Analysis of neuronal compartments has revealed many state-dependent changes in geometry but establishing synapse-specific mechanisms at the nanoscale has proven elusive. We co-expressed channelrhodopsin2-GFP and mAPEX2 in a subset of hippocampal CA3 neurons and used trains of light to induce late-phase long-term potentiation (L-LTP) in area CA1. L-LTP was shown to be specific to the labeled axons by severing CA3 inputs, which prevented back-propagating recruitment of unlabeled axons. Membrane-associated mAPEX2 tolerated microwave-enhanced chemical fixation and drove tyramide signal amplification to deposit Alexa Fluor dyes in the light-activated axons. Subsequent post-embedding immunogold labeling resulted in outstanding ultrastructure and clear distinctions between labeled (activated), and unlabeled axons without obscuring subcellular organelles. The gold-labeled axons in potentiated slices were reconstructed through serial section electron microscopy; presynaptic vesicles and other constituents could be quantified unambiguously. The genetic specification, reliable physiology, and compatibility with established methods for ultrastructural preservation make this an ideal approach to link synapse ultrastructure and function in intact circuits.

Multiplexed Superresolution CRISPR Imaging of Chromatin in Living Cells

Multiplexed Superresolution CRISPR Imaging of Chromatin in Living Cells