Novel Tools for Targeted Multicolor Imaging in the near-infrared range

On the left: 3 color imaging of tumor mass, lympathic vessels and vasculature in a tumor mouse model. On the right:multicolor flourescence guided surgery of tumor and inguinal lymph node.

HPC Bioengineers develop the first clinically adaptable targeted SWIR dyes for fast multiplexed imaging.

The context and the problem. Visualizing biological processes in their native environments is a key endeavor in modern biology.  While multiplexed fluorescence imaging is routinely used for microscopic analyses in vitro, it becomes increasingly challenging in live tissue due to interfering factors such as autofluorescence and scattering.
While intravital microscopy using two-photon excitation enables multicolor imaging of tissue sections in living animals, it still requires invasive measures and therefore, is not suitable for whole body imaging.
Recently, near infrared (NIR) imaging has emerged as a viable solution for this issue and approaches in the so-called shortwave-Infrared (SWIR)  range have demonstrated their potential for high-resolution imaging in bulk tissue with significant improvements over NIR in terms of both resolution and depth.
However, a critical bottleneck in this field, hampering its clinical translation, is the lack of targeted, biocompatible fluorescent probes suitable for the SWIR window.

The solution. An international team headed by Oliver Bruns (HPC) and Martin J. Schnermann (Center for Cancer Research, National Cancer Institute, USA) now developed a first set of promising, clinically applicable targeted SWIR dyes enabling fast multiplexed SWIR imaging in vivo. Initiated by rational computational design, the study published in Nature Methods reports on two novel classes of targeted nonamethine indocyanine dyes with significant emission in the SWIR range that can be used in combination with clinically applied ICG and IR-800CW dyes.

The results. The team developed two compounds, FNIR-872 and FNIR-1072, and thorough analysis of their spectral and photophysical properties, both dyes revealed chemical- and photostability in serum comparable to the clinically used IR-800CW dye.
Using a dedicated, custom-built imaging system for multicolor real-time imaging that incorporates three different NIR laser excitation sources (one for each of the three dyes as well as a 1300 nm LED for reflectance imaging), the authors demonstrated the potential of both dyes for multiplexing with minimal crosstalk. Importantly, both dyes proved suitable for targeted tumor imaging, comparable to the clinically used IR-800CW-mAb, with a clear capacity to delineate the bulk tumor, surrounding lymphatic vessels and tumor vasculature in multiple mouse tumor models. As this new imaging approach is not sensitive to visible light, it also allows for fluorescence-guided surgery with room lighting on, thus simplifying current workflows, which may require dimming of the room and surgical lighting for example.

The future. “These two novel conjugatable SWIR dyes open the door to multiple applications in the field of targeted multiplexed imaging, since they can be easily conjugate to multiple probes eg antibodies to visualize specifically biological structures of interest, in vivo” explains Mara Saccomano, one of the first authors of this work.
Collectively, these advances in dye chemistry and instrumentation enable simultaneous visualization of bulk tissue, fine structures, and biological processes during procedures requiring video-rate acquisition, i.e. fluorescence-guided surgery, identification of sentinel lymph nodes or perfusion measurments.
Thus, this study provides a major step forward in extending our toolbox for multiplexed imaging in various settings, paving the way to visualize so far inaccessible processes and structures.

Link to publication