Image Credit: shutterstock.com/ GiroScience
Surface-enhanced Raman scattering (SERS) nanoparticles are setting themselves to be an attractive alternative to fluorescent probes in biological labeling applications, due to their photostability and multiplexing capabilities
Yet, to realize their full potential, they need to overcome a few hurdles and a team of Researchers from the United States of America have now released a study looking at the ability of SERS nanoparticles to monitor cell dynamics and attempting to shed some light on how the field should move forward.
Studying the dynamics and evolution of biomarkers at the single cell level can help with the understanding of cancer cell proliferation, especially breast cancer. Currently, fluorescent probes are used to understanding such dynamics.
However, the next stage of this research field is to understand how the dynamics of the biomarkers evolve using a group of cells, over multiple cell generations, in an effort to isolate the most aggressive cancer stem cells. For this to occur, the biomarkers require two things- a high multiplexing ability and high photostability.
However, the issue is, currently technology using fluorescent probes is prone to photobleaching and spectral crosstalk that limits multiplexing. Therefore, it is not viable for the next phase of breast cancer stem cell research and a new alternative is required.
One area which shows great promise, is nanoparticles. In particular, surface-enhanced Raman scattering (SERS) nanoparticles. SERS have been used to image both fixed and live cells, but current research doesn’t take into account many factors. This includes distinguishing clear advantages over fluorescence labeling and the natural interaction affinity between cells and nanoparticles. The affinity also relies strongly on the various properties of the nanoparticles, including their shape size and surface properties.
The Researchers have used SERS nanoparticles to target HER2 and CD44 proteins found within breast cancer cells. The Researchers cultured the cancer cells, proliferated them and immune-stained the samples with the SERS nanoparticles. Staining with fluorescent probes was also undertaken as a control.
The fluorescence imaging was performed using a Delta Vision deconvolution fluorescence microscope (Personal Deltavision, GE Life Science) and Raman hyper-spectral images were produced using a home-built line-scanning Raman microscope- consisting of a CW laser (Serval Plus, Sacher-Laser), a cylindrical lens (Thorlabs), an entrance slit (Princeton Instruments), an oil immersion objective (Olympus), a motorized stage (PRIOR Scientific), a piezo objective scanner (PI), a CCD camera (CoolSnap EZ, Photometric) and a dark-field (Leica DM-IRM) illuminator and reflector.
The Researchers also used a combination of flow cytometry (BD LSRFortessa Cell Analyzer, BD Biosciences), dynamic light scattering (DLS, Zetatrac light scattering instrument, Microtrac, Inc), ultraviolet-visible spectroscopy (UV-Vis, Varian Cary 50 Bio, Agilent technologies), ECL Western blotting (Amersham Biosciences) and dot blotting (Amershame Biosciences) techniques throughout their analyzes.
Interested in Instrumentation? Request More Information
The Researchers were able to label both HER2 and CD44 in fixed cancer cells with a high degree of specificity, and their results correlate well with the studies produced using fluorescent probes. The SERS nanoparticles were also found to operate with a high degree of multiplexity and photostability.
The mechanism of detection was found to completely differ from the mechanism using fluorescence probes. Upon labeling of the live cells, the nanoparticles were taken up by the cells and became compartmentalized into different cellular regions. In contrast, the use of fluorescence probes develops fluorescent antibody conjugates.
The uptake rate of nanoparticles into the cells was found to be very efficient and occurred at a very fast rate. This resulted in a label distribution that is unique and does not resemble those obtained from imaging fluorescent conjugates. However, the Raman imaging of the SERS nanoparticles was shown to be of the same quality as fluorescence imaging.
As it turns out, the nanoparticles, for multiple reasons, were not suitable as contrast agents, but they were found to be outstanding candidates for flow cytometry applications. It was also found that SERS nanoparticles are not suitable for long term, live-cell monitoring of biomarker dynamics, due to the variation in their size and shape. However, given their excellent uptake rate, they have been found to excellent candidates for both in-vivo imaging applications and as non-targeted endocytic tracers.
The nanoparticles have been found to offer fantastic opportunities for imaging and sensing applications. And whilst they show great promise, great care will have to be taken to mediate the cell-nanoparticle interaction to take full advantage of their properties.
Sources and Further Reading
“Nanoparticles for live cell microscopy: A surface-enhanced Raman scattering perspective”- Navas-Moreno M., et al, Scientific Reports, 2017, DOI:10.1038/s41598-017-04066-0