Near-infrared fluorescent proteins for live cell and animal imaging

Unmet Need

Fluorescent microscopy is one of the most common research tools used by biologists and has a global market value expected to exceed $768 million by 2023. Many of the currently available fluorescent protein tags for use in cell-based assays and whole animal imaging utilize proteins that are excited using light in the visible spectrum (380-700 nm). Fluorescent proteins that are excited in the visible spectrum can lead to issues associated with cellular autofluorescence and photobleaching that contribute to experimental noise. Due to the high potential for background noise with this approach, there is a need for biologically compatible fluorescent proteins that are excited outside of the visible light spectrum.

Technology

Duke inventors have developed a series of fluorescent proteins isolated from tree frogs that are excited by far right light and emit near-infrared (NIR) light. This is intended to be used in research settings to tag proteins for visualization in both cell microscopy and whole-animal assays. Because these proteins emit light outside of the visible spectrum, there is limited background fluorescence and high compatibility with visible spectrum fluorescent proteins such as GFP. These proteins are perfectly suitable for in vivo imaging because both excitation and emission spectra lie in the ‘transparency optical window’ (650-900 nm), a region of the electromagnetic spectrum where there is low cellular autofluorescence, minimal absorption from hemoglobin and melanin, and reduced scattering. This tool is widely applicable to fluorescence microscopy techniques and can be utilized in visualization through both epifluorescence and confocal microscopy approaches. Specifically, this class of proteins, called biliverdin binding serpins (BBSs), becomes excited by NIR light once they bind to the green pigment biliverdin which is a common degradation product of heme in animals. BBSs have been demonstrated to be an effective fluorescent tool for visualization in vitro in cells and in multiple tissue types in vivo and unlocks a whole new source of diverse fluorescent templates.

Other Applications

As a research tool this technology could also be used to develop a molecular photo switch for super resolution imaging techniques due to the reversible photobleaching effect of the reversible biliverdin binding.

In the healthcare setting, BBSs could be utilized to trace and localize sites of hemorrhages due to their high affinity for the heme catabolic product biliverdin.

This has applications in detecting vesicle occlusions, intraplaque hemorrhages, and heme degradation in atherosclerotic lesions.

BBSs are also compatible with photoacoustics imaging at the whole-body scale and can be applied to image vascular burdens, plaque formation, and angiogenesis at sites of early tumor growth.

Advantages

  • Minimized autofluorescence with NIR light compared to GFP
  • Distinct excitation NIR wavelengths allow for co-imaging with fluorophores in visible light spectrum
  • Improves upon vertebrate research compatibility of other bacteria-derived NIR-excitable proteins due to isolation from tree frogs
  • Highly stable in large pH range
  • High effective brightness
  • Shelf-stable for over 2 months