Multiplexed Raman molecular imaging

A staining formulation, useful for staining a sample to identify at least one chemical moiety on at least one surface of the sample, wherein:

• the staining formulation comprises at least two SERS-flavors;
• each SERS-flavor comprises at least one surface enhanced Raman spectroscopy nanoparticle (SERS-NP);
• each SERS-NP comprises a Raman reporter and a Raman active core;
• each SERS-flavor's at least one SERS-NP comprises a Raman reporter and/or Raman active core that is different than those of the other SERS-flavors in the staining formulation;
• each SERS-flavor has a Raman spectrum determined at an excitation wave with about 785 nm wavelength;
• the staining formulation has a value of a log(det) in a range of a value less than or equal to log(det)most to a value greater than or equal to 95% of log(det)least;
• the log(det) is calculated by using a spectral dissimilarity objective (SDO) and a singular value decomposition (SVD) for a Raman spectrum of the staining formulation determined at an excitation wave with about 785 nm wavelength;
• log(det)most is most preferable log(det);
• log(det)least is least preferable log(det);
• log(det)most is calculated by using the equation, log(det)most=−0.0105X2−0.0861X−0.1917;
• log(det)least is calculated by using the equation log(det)least=−0.2032X2+0.1229;
• and
• X is number of SERS-flavors.

A SERS-flavor comprising a surface enhanced Raman spectroscopy nanoparticle (SERS-NP) wherein:

• the SERS-NP comprises:
• a Raman active core and a Raman active layer;
• or
• a Raman active core, a Raman active layer, and a labeling agent;
• or
• a Raman active core, a Raman active layer, a labeling agent, and a shell;
• the Raman active core has an outer surface;
• the shell has an inner surface and an outer surface;
• the Raman active layer is positioned between the outer surface of the Raman active core and the inner surface of the shell;
• the labeling agent is attached to the Raman active layer or the outer surface of the shell;
• the Raman active layer comprises a Raman reporter;
• the Raman active core and the Raman reporter enhance Raman scattering and are thereby suitable for surface-enhanced Raman spectroscopy (SERS);
• and
• the Raman reporter is selected from Table 9, Raman reporter IDs 4-6, 8, 24, 29-31, 34, 39-43, and 46-49, or a combination thereof.