Vishwas N. Joshi

Large Covalently Linked Fluorescent and Gold Nanoparticle Immunoprobes

FluoroNanogold™ probes containing both a fluorescent label and the Nanogold® cluster covalently linked to a targeting molecule have been successfully used for correlative fluorescence and electron microscopy (EM) [1]. However, Nanogold is small (1.4 nm) and requires silver or gold enhancement for visualization [2]. This produces much greater size variability than is found in larger colloidal gold preparations. Silver enhancement may lead to non-specific background; and silver can be chemically etched by osmium tetroxide (OsO4) fixation leading to signal loss [2]. We have prepared covalently linked combined fluorescent and larger gold nanoparticle probes for correlative microscopy that can be directly visualized by EM without the need for autometallographic enhancement. Gold nanoparticles, 2 nm to 40 nm in diameter, were stabilized and functionalized using a self-assembling coating comprising a hydrophobic chelating thiol domain to seal the gold surface from aqueous media, and hydrophilic terminal functional groups (polyethylene glycol, PEG) that biocompatibilize the gold nanoparticles and enable site-specific covalent probe conjugation. This provides high stability and a wide choice of surface properties and conjugation reactions. 5 nm gold nanoparticles were prepared by direct reduction of aqueous solution of gold (III) salt with sodium borohydride in the presence of a mixture of solubilizing non-functional and protected amine-functionalized thiols, followed by purification using sucrose density gradient (10–30%) ultracentrifugation (Ti-41 rotor, 26,000 rpm, 45 min, 5 °C; the intense red band that travels ~ 4 cm from the top contains 5 nm gold). Amine-functionalized gold particles were prepared by deprotection in methanolic hydrochloric acid. Specific reactivity was demonstrated by reaction with commercial N-hydroxy succinimido-(NHS) Cy-5.5 fluorescent dye. This conjugate was further purified by Superose-12 size exclusion chromatography (Figure 1). Unstabilized particles of this size are precipitated by 0.1 M sodium chloride, but the coated particles remained dispersed and suspended, even in 1.0 M sodium chloride solution. They were repeatedly centrifuged and completely resuspended without leaving any solid precipitate behind; identical treatment of conventional protein or macromolecule-stabilized colloidal gold conjugates always results in loss of a fraction as an insoluble pellet that cannot be resuspended. Figure 1 UV-visible, fluorescence (Exλ 660 nm) spectra, and TEM (a, b, and c) of fluorescent 5 nm gold-Cy-5.5 conjugate purified by density gradient ultra-centrifugation (d) followed by size exclusion chromatography (vide supra). Combined 5 nm gold-secondary antibody-Alexa Fluor 594 conjugates were prepared by covalently linking NHS- and Maleimido- activated 5 nm gold particles to the secondary antibody molecule, IgG and F(ab′) respectively. NHS-modified Alexa Fluor 594 dye was then reacted with the conjugate. The product was purified using sucrose density gradient ultracentrifugation followed by gel filtration as above. The anti-rabbit-F(ab′) conjugate was used as a secondary probe against a polyclonal rabbit anti-red blood cell antibody to label sheep red blood cells in suspension and the labeling was observed by fluorescence microscopy using Nikon G-2A filter set (Figure 2). The anti-mouse-IgG conjugate was used as a secondary probe with mouse anti-human AE1/AE3 primary that produced clear staining of cytokeratin in human tonsil tissue (Figure 3). The relative quantum yields for the fluorescent gold conjugates were 22–35% of the corresponding commercial dye labeled secondary antibodies. Figure 2 Bright field (A) and fluorescence images (B) of sheep red blood cells labeled with 5 nm gold anti-rabbit-F(ab′)-Alexa Fluor 594; C and D are images for control cells in which the primary rabbit anti-sheep red blood cell antibody was excluded. Figure 3 Fluorescence (A), bright field (B), and bright field image following silver development (C) of cytokeratin stained tonsil tissue with mouse anti-human AE1/AE3 primary and 5 nm gold anti-mouse-IgG-Alexa Fluor 594 secondary antibody; D is corresponding ...

EnzMet for Versatile, Highly Sensitive Light and Electron Microscopy Staining

EnzMet (enzyme metallography) is a staining and detection method comprising a peroxidase probe developed with a metallographic silver substrate. The dense, black signal is readily observed by light microscopy, and the electron-dense, particulate product clearly visualized in the electron microscope (EM) [1]. Background is virtually zero and diffusion is negligible. EnzMet gives clear enumeration of gene copies in in situ hybridization [2] and highly differentiated, permanent staining in immunohistochemistry [3]. Because organic chromogens such as diaminobenzidine (DAB) are used mostly with contrast enhancers such as nickel (II) [4], or with polymerized peroxidase probes to amplify the signal [5], a comparison with these methods was undertaken. Paraffin-embedded bladder carcinoma sections from the same series were stained in parallel using AE1/AE3 primary monoclonal antibody and peroxidase secondary. EnzMet using a monomeric peroxidase probe was compared with standard DAB and with DAB with Ni(II) enhancement using the same probe, and with DAB using a polymerized secondary probe (Envision, Dako). EnzMet gave higher sensitivity and contrast than others (Figure 1), and comparable results with up to 50-fold lower primary antibody concentrations.

Correlative FRET: new method improves rigor and reproducibility in determining distances within synaptic nanoscale architecture

A new correlative Förster Resonance Energy Transfer (FRET) microscopy method using FluoroNanogold™, a fluorescent immunoprobe with a covalently attached Nanogold® particle (1.4nm Au), overcomes resolution limitations in determining distances within synaptic nanoscale architecture. FRET by acceptor photobleaching has long been used as a method to increase fluorescence resolution. The transfer of energy from a donor to an acceptor generally occurs between 10-100Å, which is the relative distance between the donor molecule and the acceptor molecule. For the correlative FRET microscopy method using FluoroNanogold™, we immuno-labeled GFP-tagged-HeLa-expressing Connexin 35 (Cx35) with anti-GFP and with anti-Cx35/36 antibodies, and then photo-bleached the Cx before processing the sample for electron microscopic imaging. Preliminary studies reveal the use of Alexa Fluor® 594 FluoroNanogold™ slightly increases FRET distance to 70Å, in contrast to the 62.5Å using AlexaFluor 594®. Preliminary studies also show that using a FluoroNanogold™ probe inhibits photobleaching. After one photobleaching session, Alexa Fluor 594® fluorescence dropped to 19% of its original fluorescence; in contrast, after one photobleaching session, Alexa Fluor 594® FluoroNanogold™ fluorescence dropped to 53% of its original intensity. This result confirms that Alexa Fluor 594® FluoroNanogold™ is a much better donor probe than is Alexa Fluor 594®. The new method (a) creates a double confirmation method in determining structure and orientation of synaptic architecture, (b) allows development of a two-dimensional in vitro model to be used for precise testing of multiple parameters, and (c) increases throughput. Future work will include development of FluoroNanogold™ probes with different sizes of gold for additional correlative microscopy studies.

Development and characterization of nanoparticles as imaging probes for correlative optical and electron microscopy

Nanoparticles of several different compositions and lattice structures that have been characterized by transmission electron microscopy, scanning/transmission electron microscopy, X-ray spectroscopic (EDX) analysis, and diffraction patterns may be conjugated to biological targeting agents for use as labels that can localize and differentiate multiple targets at nanometer resolution in complex biological specimens. Small (most 0.8–3.0 nm) particles composed of iron oxide/carbon, gold, iridium and bismuth/silica may be differentiated by both EDX and diffraction patterns and offer the following advantages over conventional colloidal gold labeling: smaller probe size and faster specimen penetration, higher labeling precision, higher, more consistent labeling density, and nearer quantitative labeling. The combination of small probe size and number of compositions available affords increased multiplexing capability.

Statistical Design of Experiments to Ensure “Rigor and Reproducibility” in Imaging Sciences

The most common approach to experimental design employed by many scientists today is OneVariable-At-a-Time (OVAT), in which one variable at a time is changed, keeping all other variables in the experiment fixed (or constant); then, the change in the resulting outcome is observed. Quantifiable outcomes of the experiments are often measured using scientific instruments or equipment, e.g., in spectroscopic and microscopic techniques. The variations observed in the outcome of an experiment may stem from two sources: a) it may be a direct consequence of the intentional change in the input variable, usually the case if the outcome variable is correlated with the changing input variable; or b) it may be an error or variation in the measurement system (Fig. 1). Drawing correct logical conclusions when simultaneously working with nanoscale materials and devices and operating the measurement equipment near or at its performance limit, requires appropriate quantification of all sources of variability.

Biocompatible, Biodegradable Radio-opaque Polymer Nanoparticles.

The conventional molecular iodine X-ray contrast agents (ICAs) clear rapidly through kidneys and by extravascular diffusion allowing only short imaging times [1]. To address this larger-sized ICAs based on liposomes, polymers, micelles, dendrimers with polyethylene glycol (PEG) core, and metal nanoparticles with longer blood half-lives, or blood-pool agents, that enable targeting and temporal imaging have been developed [2]. Despite varying degrees of success with regards to higher contrast, lower osmolality, longer circulation times and target-specific imaging with these experimental agents the issues related to instability in the physiological medium, toxicity, accumulation in liver and spleen, long-term whole body retention and overall performance remain to be solved [2].

Gold Nanoparticle Photoaffinity Labels for Electron Microscopy

The conventional methods for steric stabilization of transition metal and semiconductor nanoparticles (NPs) involve the use of organic or natural polymers, surfactants, lipid bilayers and silica coatings. These methods significantly increase overall size of the NPs [1], and that could be problematic for some applications because of dielectric nature of the coating versus the conducting or semiconducting properties of the metal core may affect the properties and performance of the stabilized NPs in the desired end-application. To reduce overall size of the stabilized NPs we have developed a selfassembling coating comprising a hydrophobic metal chelating domain that seals the metal surface from aqueous buffers and a variable length hydrophilic terminal groups that stabilize and biocompatibilize the NPs (Figure 1). The terminal groups also provide means to further functionalize and cross-link NPs to specific groups when a small proportion (10-30%) of activatable terminal groups, e.g., -NH2 or -COOH, are included on the NP surface. We have used this strategy to develop metal NP labels for microscopic localization and imaging [2,3]. We now report gold nanoparticle (AuNP) photo-affinity labels (PALs) prepared following a similar strategy. PALs enable direct verification of the spatial proximity of macromolecular components of proteins that are not amenable to crystallography or NMR [4]; the smaller-sized heavy metal NP labels will enable unambiguous localization macromolecular components by electron microscopy and tomography at higher resolution.

Gold Nanoparticle Technology to Address Variability in EM Labeling

Conventionally, colloidal gold labeled antibodies have been the method of choice for electron microscopic labeling, but suffer from limitations. Reliable conjugation protocols exist only for antibodies and a few proteins and require additional macromolecules for stabilization: these yield large probes that penetrate slowly and may only label a small fraction of closely spaced targets. When used as secondary probes especially, the “radius of uncertainty,” or distance from the binding site to the gold label, may be as large as 15-25 nm or more, prohibiting the differentiation of discrete targets in small structures such as synapses. In addition, multiplexing is traditionally achieved by the use of labels of different sizes. Because each preparation of colloidal gold contains a range of sizes, multiplexing by size is generally limited to two or three targets. Furthermore, differences in probe size, which limits antigen access and labeling density, can mean that labeling results may not reflect the relative abundance of the different targets.

Luminescent Ruthenium Complex Labels for Correlative Microscopy.

Biological processes are characterized both by their distribution where they occur in tissues, cells and organelles and by the structure and interaction of their components. Diffraction-limited light microscopy (LM) and “super-resolution” microscopy (SRM) enable observation of lives cells and tissue details down to hundreds of μm and tens of nm, respectively [1]. Electron microscopy (EM) on the other hand provides much higher resolution [2]. The combination of LM with EM or correlative light and electron microscopy (CLEM), therefore, is a powerful method for correlating dynamic functional information from LM/SRM with static, high-resolution structural information from EM [3].

Combined Texas Red and 1.8 nm FluoroNanohold™ for Multimodal Imaging

* Nanoprobes, Incorporated, 95 Horseblock Road, Unit 1, Yaphank, NY 11980 ** Dept. of Physics and Astronomy, Stony Brook University, Stony Brook, NY 11794 *** Dept. of Materials Science and Engineering, Stony Brook University, Stony Brook, NY 11794 **** Dept. of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794 ¶ Present address: Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, CA 94025 † Present address: Dept. of Physics and Astronomy, Northwestern University, Evanston, IL 60208 and Argonne National Laboratory, Argonne, IL 640439