September 2016 Jeremy Grant Multiplexed SERS imaging in biological systems using biocompatible Raman active Nanostars September 2016 Jeremy Grant The ability to simultaneously image a large number of spectrally distinct species could transform how we study biological systems.

Fluorescence Imaging Issues For complex biological systems we would like to map many different species at the same time. 1)In order to get sufficient count rates (take images fast) we turn up the laser power, this results in data which is hard to fit due to blinking and bleaching. Only a small number of fluorophores can be distinguished simultaneously (2-5). Limits the number of features that can be spatially correlated.

Raman Spectroscopy Inelastic Scattering – Energy of incident photon changes, a little. Molecule E + Evib E - Evib E Laser A Raman spectrum is essentially a molecular vibrational “Fingerprint”. Spectral linewidths are 100-1000 x narrower than fluorescence (Multiplexing) Unfortunately, Raman efficiency is 1014 –1016 times weaker than fluorescence

Surface Enhanced Raman Spectroscopy (SERS) Signal Enhancements as large as 1010 –1012 near metal surface Electric field enhancement + chemical surface effect For Nanoparticles enhancement drops off rapidly with distance: Only those molecules at the nanoparticle surface are enhanced. So No enhancement in the rest of the sample (reduced Raman background)

Nanospheres Narrow linewidths – Probes can be easily identified Stable signal – no blinking, not environmentally sensitive 545nm 580nm Napthalene thiol Mercaptopyridine BPE Challenges (Problems) Signal – spherical core geometries typically under perform (low signal).

Newest Geometry –Nano Stars Raman NanoStars generate stronger signals than fluorophores when imaged in a “traditional” confocal microscope! FL Raman Allgeyer, E., Browne, M., Pongan, A., Mason, M. “Signal Comparison of Single Fluorescent Molecules and Raman Active Gold Nanostars” Nano Letters 9(11), 2009, 3616-3819. This NEW system has the promise of overcoming previous signal limitations!!

Good Biostability, much easier chemistry (though not trivial). PEG instead of glass Poly Ethylene Glycol Dithiol Raman Tags are added via thiol bonding just prior to PEGylation. Good Biostability, much easier chemistry (though not trivial).

Specific Aim 1 Synthesize and characterize up to 10 Raman active nanostar probes for use in spectral multiplexing. 1b) Use ~12 nm spherical cores to produce ~25, 50 nm nanostars in DMF (modified Liz-Marzan method, 2010) 1a) Synthesize ~12, 25, 50 nm gold Raman spherical cores using modified Turkevich (1951) method. 1c) Investigate aqueous nanostar chemistry to eliminate DMF toxicity (Jianping Xie , 2007) 1d) Perform surface passivation (PEG) and modification with Raman reporters. 1e) Perform probe composition optimization using in vitro spectral characterization.

Specific Aim 2 Develop spectroscopic scanning and spectral multiplexing Raman imaging software. 2a) Optimize Raman system hardware and software for appropriate signal levels. 2b) Select and optimize Raman signal analysis algorithm.

Specific Aim 3 Application of Raman nanostar and Raman imaging technologies to cellular uptake. 3a) Determine the relationship between nanoparticle size and geometry and passive uptake. 3b) Perform bulk determination of “typical” Raman signal levels in vivo (tissue culture)