Supplement 1: Fractal propagation method enables realistic optical microscopy simulations in biological tissues

Supplemental document. Originally published in Optica on 20 August 2016 (optica-3-8-861

Visualization 1: Fractal propagation method enables realistic optical microscopy simulations in biological tissues

Propagation of Gaussian beam through fractal tissue model. Originally published in Optica on 20 August 2016 (optica-3-8-861

Surface-enhanced Raman scattering (SERS) nanoparticles.

<p>(a) Multiple flavors of nanoparticles exist where each nanoparticle contains a gold core coated with a Raman-active layer, encased in a silica shell. (b) Raman spectra of five nanoparticle flavors. (c) Example result from a least-squares routine showing the ability to demultiplex two different nanoparticles from a mixture under noisy conditions.</p

Method for Assessing the Reliability of Molecular Diagnostics Based on Multiplexed SERS-Coded Nanoparticles

<div><p>Surface-enhanced Raman scattering (SERS) nanoparticles have been engineered to generate unique fingerprint spectra and are potentially useful as bright contrast agents for molecular diagnostics. One promising strategy for biomedical diagnostics and imaging is to functionalize various particle types (“flavors”), each emitting a unique spectral signature, to target a large multiplexed panel of molecular biomarkers. While SERS particles emit narrow spectral features that allow them to be easily separable under ideal conditions, the presence of competing noise sources and background signals such as detector noise, laser background, and autofluorescence confounds the reliability of demultiplexing algorithms. Results obtained during time-constrained <i>in vivo</i> imaging experiments may not be reproducible or accurate. Therefore, our goal is to provide experimentalists with a metric that may be monitored to enforce a desired bound on accuracy within a user-defined confidence level. We have defined a spectral reliability index (SRI), based on the output of a direct classical least-squares (DCLS) demultiplexing routine, which provides a measure of the reliability of the computed nanoparticle concentrations and ratios. We present simulations and experiments to demonstrate the feasibility of this strategy, which can potentially be utilized for a range of instruments and biomedical applications involving multiplexed SERS nanoparticles.</p></div

Spectral quality characterization of two-flavor mixtures.

<p>(a) Composite errors for multi-particle mixtures are gamma-distributed. Vertical lines indicate bounds for the 80^th percentile of error values (<i>e</i>₈₀) that may occur for a particular SRI. (b) A plot of error (<i>e</i>₈₀) vs. SRI for a 1∶1 mixture of particle flavors and a 5∶1 mixture of flavors. Simulations (solid lines) closely predict experimental results. (c) A plot of the minimum SRI required to ensure a composite error ≤10% with 80% confidence. This dual-flavor example shows how the minimum SRI value depends upon the mixture ratio.</p

Spectral quality characterization of three-flavor mixtures.

<p>The plot shows percent error (<i>e</i>₈₀) in the ratio between particle flavors as a function of SRI.</p

Visualization 2: Fractal propagation method enables realistic optical microscopy simulations in biological tissues

Propagation of Bessel beam through fractal tissue model. Originally published in Optica on 20 August 2016 (optica-3-8-861

Measurement apparatus.

<p>(a) A spectrometer with CCD detector is used to capture Raman signals from a nanoparticle sample illuminated with a 785-nm laser source. See text for details. (b) Example of a strong signal with a high SRI and (c) a weak signal with a lower SRI, in which noise and broadband background signals increasingly dominate over the SERS signals. Representative SERS peaks are numbered 1–4 and the peak of the broadband background is labeled with a star.</p

Algorithm summary.

<p>A flowchart illustrating how SRI may be used in practice to ensure that a SERS-based multiplexed molecular diagnostic is reliable. See text for details.</p

REMI approach performed on a human breast tissue specimen stained with a 5-flavor NP mixture (EGFR-, HER2-, CD24-, CD44-, and isotype-NPs, 150 pM/flavor).

<p>(<b>A</b>) Ratiometric images of a human breast tissue specimen. From top to bottom, the rows display ratiometric images of EGFR/isotype-NP, HER2/isotype-NP, CD24/isotype-NP and CD44/isotype-NP. From left to right, the columns display ratiometric images obtained with a decreasing number of spectral channels. (<b>B</b>) A photograph of the tissue specimen. (<b>C</b>) H&E histology of the specimen, with higher magnification views of fat (left), normal breast tissue (middle), and tumor (right). Unlabeled scale bars represent 200 μm. (<b>D</b>) Average error (%) in the measured NP ratios when using spectral compression in comparison to the gold-standard images (full 1024 spectral channels). The error bars represent the standard deviation amongst all pixels in the image.</p