Bioimaging with Macromolecular Probes Incorporating Multiple BODIPY Fluorophores

Abstract

Seven macromolecular constructs incorporating multiple borondipyrromethene (BODIPY) fluorophores along a common poly(methacrylate) backbone with decyl and oligo-(ethylene glycol) side chains were synthesized. The hydrophilic oligo(ethylene glycol) components impose solubility in aqueous environment on the overall assembly. The hydrophobic decyl chains effectively insulate the fluorophores from each other to prevent detrimental interchromophoric interactions and preserve their photophysical properties. As a result, the brightness of these multicomponent assemblies is approximately three times greater than that of a model BODIPY monomer. Such a high brightness level is maintained even after injection of the macromolecular probes in living nematodes, allowing their visualization with a significant improvement in signal-to-noise ratio, relative to the model monomer, and no cytotoxic or behavioral effects. The covalent scaffold of these macromolecular constructs also permits their subsequent conjugation to secondary antibodies. The covalent attachment of polymer and biomolecule does not hinder the targeting ability of the latter and the resulting bioconjugates can be exploited to stain the tubulin structure of model cells to enable their visualization with optimal signal-to-noise ratios. These results demonstrate that this particular structural design for the incorporation of multiple chromophores within the same covalent construct is a viable one to preserve the photophysical properties of the emissive species and enable the assembly of bioimaging probes with enhanced brightness.

Ratiometric Calcium Imaging of Individual Neurons in Behaving <em>Caenorhabditis Elegans</em>

“…Slit- or resonant point-scanning confocal microscopes typically found in shared confocal facilities also give excellent performance during ratiometric Ca 2+ imaging. Such instruments can be used to capture two or more fluorescence channels along with brightfield 24 . In this case, the confocal pinhole should be opened to its maximum diameter, and a spectral detector should be used to separate GCaMP5, mCherry, and infrared brightfield signals.…”

“…Inject GCaMP5- and mCherry-expressing plasmids along with the pL15EK visible rescue marker plasmid into the gonads of LX1832 lite-1(ce314), lin-15(n765ts) X mutant animals and recover non-Muv lin-15(+) animals expressing GCaMP5 and mCherry from a high-copy transgene 20 21 22 . Use the lite-1 mutant background to reduce blue-light avoidance behavior 23 24 .…”

Fluorophore Multimerization as an Efficient Approach towards Bright Protein Labels

“…A significant effort in the field was done to develop bright staining reagents by employing multimerization of smallmolecule fluorophores on non-fluorescent polymeric backbones such as polymethacrylate derivatives, [113] copolymers of Nacryloylmorpholine and N-acryloxysuccinimide (poly(NAM-co-NAS)), [114] and many others [115][116][117] (Figure 6, Table 6). The requirements to these dyes are often similar to those of π-conjugated polymers, e. g. high water-solubility, presence of bioconjugation [a] M n .…”

“…The authors synthesized multimers with up to 7.4 dyes per polymer and with a brightness three times higher relative to the monomeric dye and demonstrated selective binding of the resulting conjugates to their targets. [115] Zhang et al described water-soluble fluorescent oligoethylenglycol derivatives of poly (methacrylate) decorated with rhodamine. [113] The-authors used Herceptin antibody with site-directed conjugation of polymerization initiator enabling an in situ fluorophore labelling.…”

Fluorescent Copolymers for Bacterial Bioimaging and Viability Detection

“…4,29 Furthermore, these polymeric materials can now be functionalized in order to be more biocompatible and water-soluble, making them suitable for multi-color bioimaging uses. 30 The combination of these advanced features has made fluorescent polymeric materials an important and highly promising tool, either for fluorescence imaging and sensing, or for in vitro and in vivo 31 labeling of bacteria, cells, 17 tissues, and whole organisms.…”