2.1.

Experimental Reagents

SERS-active gold NPs were purchased from Cabot Security Materials Inc. (Mountain View, California) and consist of a 60 nm colloidal gold core, an adsorbed layer of the Raman-active molecules, and a 30 nm thick thiolated silica encapsulant layer that stabilizes the reporter molecule on the AuNP surface and provides a reactive substrate for bioconjugation steps. The four reporter molecules used were S420 (4,4’-dipyridyl), S421 (d8-4,4’-dipyridyl), S440 (trans-1,2-Bis(4-pyridyl)-ethylene), and S481(4-Azobis(pyridine)). Peaks selected for imaging were located at $1295{\mathrm{cm}}^{-1}$(S420), $1578{\mathrm{cm}}^{-1}$(S421), $1339{\mathrm{cm}}^{-1}$(S440), $1164{\mathrm{cm}}^{-1}$(S481).

2.2.

Imaging System Design

The widefield Raman imaging system was designed for small animal imaging and consists of a high-power, single transverse mode laser source (CleanLaze 785, BWTek, Newark, Delaware) which is collimated and reflected onto the sample stage by a dichroic mirror (RazorEdge, Semrock, Rochester New York). Scattered light is collected by an objective and relay lens arrangement and laser light is removed by a notch filter (StopLine, Semrock) prior to entering the tunable filter module. The tunable filter is comprised of bandpass filters designed for operation at non-normal incidence (Versachrome $900/11$, Semrock), which are offset so as to produce a transmission FWHM of approximately 4 nm from 810 to 890 nm. One of the filters was requested from red-shifted stock so as to maximize the tuning range of the composite design (to minimize the broadening of FWHM at near-normal angles of incidence). The filters are mounted on a precision encoded rotary stage (PRM1Z8, Thorlabs, Newton New Jersey), which has a bidirectional repeatability of $\pm 0.1\mathrm{deg}$ which is equivalent to approximately $3.95{\mathrm{cm}}^{-1}$ in Raman shift. The filtered light is imaged onto a cooled electron multiplying charge coupled device (EMCCD) (iXon DV885, Andor, Belfast United Kingdom) which is air cooled to $-70°\mathrm{C}$ and operated in baseline-clamped mode. The field of view of the system was $2.2{\mathrm{cm}}^2$ at a working distance of 12 cm, with a pixel resolution of 50 µm.

2.3.

Raman Image Processing

As previously described,²² background removal was applied to all Raman images in a standardized fashion; images were acquired at spectral locations immediately blue/red shifted from the peak of interest in which there was no expected spectral components from any of the other SERS reporter molecules. The change in intensity between the two reference images was assumed to be linear and an estimated background signal level was interpolated on a per-pixel basis and subtracted from the measured peak image. In the case of multiplex experiments, images were also corrected by a compensation matrix to account for inter-reporter signal overlap. The compensation values were determined by imaging pure reporter molecule solutions in each bandpass channel and were fixed for each particular bandpass definition set—because the reporter spectra were stable over time, the compensation was only calculated once and applied automatically for all later imaging sessions.

As certain key determinants of image intensity (such as working distance and CCD gain) were not absolutely calibrated in the system due to the components used, the images produced cannot be quantified in terms of an absolute number of photons/solid angle/unit time. Images are displayed scaled by the integration time to present a relative number counts for a given instrument setup, similar to what is done with comparable fluorescence imaging systems.

2.4.

Linearity, Multiplexing, Depth of Detection Imaging Assays

SERS AuNPs were diluted in water and 200 μL aliquots of each concentration were plated in triplicate in 96 well clear-walled assay plates (Sarstedt, Montreal, Quebec). Raman bandpass images were acquired in each well for each reporter peak with an integration time of 3 s, averaged five times. Intensity data was recorded from an ROI corresponding to the known laser illumination area. To estimate the depth imaging capability of the system, a capillary tube filled with a dilute solution of S421 SERS NPs in various depths of a tissue-mimicking solution (1% Intralipid) having a reduced scattering coefficient of approximately ${\mu }_s^{\prime }=1{\mathrm{mm}}^{-1}$ similar to that of soft tissue in the NIR with negligible absorption.²³ Images were acquired at each depth with an integration time of 5 s, averaged five times. The sensitivity limit was estimated as the point at which a linear fit of the signal/background curve equaled unity.

2.5.

In Vivo Raman Imaging

All animal procedures were carried out with institutional approval (University Health Network, Toronto, Canada), using 8-week old female nude mice (Taconic, Hudson, New York). SERS-active or control (reporter-free) AuNPs were diluted to the desired concentration in acrylamide monomer solution (Sigma-Aldrich, Oakville Ontario) which was then polymerized to form an optically clear matrix that prevented the diffusion of the AuNPs over time in vivo. Mice were anesthetized using a mixture of ketamine/xylazine and placed on a heated stage under the Raman imaging system. A total of 25 μL AuNP-acrylamide discs were implanted in triplicate subcutaneously along the dorsum of the mouse and imaged directly. The laser spot size and power level were adjusted to remain at or below the ANSI skin exposure limit for CW 785 nm laser light. Images were acquired with an integration time of 5 s, averaged five times.

3.1.

Nanoparticle Design

The NPs employed consisted of a colloidal gold core having a mean diameter of 60 nm with an adsorbed layer of Raman reporter molecules, followed by a 30 nm thick silica shell. The Raman spectra of the four dyes used in this study are shown in Fig. 1. The silica encapsulant ensures that there is no desorption of the dye from the colloidal core (leading to a signal decrease), nor any core–core aggregation of the gold NPs (leading to a possible signal increase) as a result of changes in the NP’s chemical environment. This physical stability ensures that SERS peak intensities measured prior to use remain unchanged once the NPs are placed in a complex biological milieu, allowing for quantitative multiplex imaging.

Fig. 1

785 nm excitation surface enhanced Raman scattering (SERS) spectra for the four reporter molecules used in this study—the peaks used to image each reporter in multiplex experiments are indicated by (*).

The relative SERS signal intensities of these NPs have been found to be stable over time in biological media, and show no signs of photobleaching/degradation over extended periods of continuous imaging.²⁴ These findings contrast starkly with fluorescence-based imaging agents whose signal can be greatly influenced by small changes in pH or through processes such as chemisorption, and where photobleaching can reduce signals to undetectable levels in only a few minutes of continuous monitoring.²⁵

3.2.

Widefield Raman Imager Design

In order to permit rapid imaging of multiple SERS peaks, it was necessary to implement a tunable filter with a sufficiently narrow bandpass to prevent cross-talk between probes, while still maintaining high overall transmission and fast switching between wavelengths of interest. The system uses excitation light at 785 nm to maximize tissue penetration in the “tissue optical window,” and at this wavelength the majority of SERS peaks from the SERS NPs used are $\sim 2\mathrm{nm}$ FWHM. Acousto-optical tunable filters (AOTFs) and liquid crystal tunable filters (LCTFs) are available with bandpass widths of $<5\mathrm{nm}$ FWHM which would be suitable for Raman imaging. However, since both designs recover only one polarization of light, when imaging diffusely scattered light in vivo their overall transmission decreases to typically $<30\%$. The cost of large-format, imaging-quality AOTFs or LCTFs with a suitably narrow bandpass for Raman imaging is generally in excess of $20,000 USD.

The design of our system is shown in Fig. 2, and is based on two relatively inexpensive offset bandpass filters which are designed to be used in spectral-tuning applications. By varying the angle of the filters with respect to the incoming light the center wavelength of transmission shifts proportionately. The offset between the filters sets the overall composite FWHM of the filter system, which is constant over a linear tuning range of 400 to $1500{\mathrm{cm}}^{-1}$ (Figs. 3 and 4) while maintaining a transmission $>90\%$. The filters are housed on an optically encoded rotation stage which has an effective positional accuracy of $3.95{\mathrm{cm}}^{-1}$ and changes between adjacent SERS peak locations in $<1\mathrm{s}$. The total cost of the two filters and rotation stage is approximately $3,000 USD.

Fig. 2

(a) Schematic diagram of widefield SERS imaging system components—the sample is illuminated by laser light from a collimator (COL) via a dichroic mirror (DM), signal is collected by an objective lens (OL), relayed through a notch filter (NF) and tunable filter (TF) via a pair of relay lenses (L1, L2) on to a charge coupled device (CCD) detector. (b) Photo of imager configured for in vivo imaging with inset image of the tunable filter module with offset bandpass filters.

Fig. 3

Measured full width half maximum (FWHM) of tunable filter as a function of center wavelength; the FWHM is fixed at approximately 4 nm from 810 to 890 nm, which corresponds to 400 to $1500{\mathrm{cm}}^{-1}$ at 785 nm excitation.

Fig. 4

Measured center wavelength of the tunable filter as a function of the angle of incidence from the optical axis. This is in good agreement with the predicted value based on the effective index of refraction for the filter.

The SERS imaging system automatically applies our previously described background minimization approach²² by capturing predefined anti-Stokes/Stokes images for each SERS peak and interpolating the pixel intensities to determine the approximate fluorescence background contribution to each peak image, which is then subtracted. Crosstalk between channels is generally $<5\%$ and can be compensated for exactly using the a priori known spectral features of each SERS dye used and the measured instrument response as a function of wavelength. The degree of crosstalk between SERS channels is a function of the filter bandpass and the inter-peak spacing of the different reporter molecules and, by selecting imaging peaks that are well separated, spectral overlap is generally reduced to $<1\%$ such that compensation is not applied.

3.3.

Widefield SERS Imaging Performance

The system response was extremely linear ($R^2=0.998$, Fig. 5) over a range of (0.01 to 100) pM of NP concentration, demonstrating a sensitivity which is two orders of magnitude better than commercial in vivo imaging systems with quantum dots.²³ At present, the limit of detection (LOD) is set by the combination of the fluorescence background signal level of the specimen and the instrument noise, which for SERS NPs diluted in a 96-well high-throughput assay plate results in an LOD less than 0.01 pM. In the case of the assay plate there is little detectable fluorescence signal and the sensitivity is primarily limited by stray light in the imaging system—the tunable filter operates by reflecting light outside its bandpass and, for certain SERS peaks, the angle of incidence of the filter may reflect off-band light from the stage enclosure and into the CCD, which quickly leads to saturation. At concentrations above 50 pM the optical density of the SERS NP solution begins to limit the amount of light detected by absorption of both the excitation laser light and the Raman scattered signal photons.

Fig. 5

Widefield Raman image intensity as a function of SERS nanoparticle (NP) concentration (S421). Triplicate 200 μL aliquots of each concentration were imaged in a clear-wall 96 well plate, error bars represent $+/-$ standard deviation of the three replicates.

As shown in Fig. 6, the system response in vivo is also extremely linear ($R^2=0.995$) and with a fixed integration time of 5 s has a LOD below 2.5 pM. At this concentration, the signal to background ratio (as compared to 40 pM reporter-free control NPs) is reduced to $1.20:1$, primarily as a result of the skin fluorescence which leads to CCD saturation prior to collecting a significant number of Raman scattered photons.

Fig. 6

(a) Widefield Raman image of 80 pM SERS reporter molecules injected subcutaneously on a nude mouse dorsum, superimposed on a white light structural image (b) Results of serial dilution of S481 measured in vivo, measured in triplicate, demonstrating a limit of detection (LOD) below 2.5 pM using an acquisition time of 5 s.

3.4.

Multiplex SERS Imaging

To demonstrate the multiplex imaging capability of the system, drops of four spectrally distinct SERS NPs were placed on filter paper and illuminated simultaneously while the system imaged at each of the four individual SERS peaks, highlighted in Fig. 1. As shown in Fig. 7, the individual dyes were easily distinguished based on their unique spectral peaks, with $<5\%$ overlap between image channels. As each SERS peak being imaged produces a different intensity (area under curve in Fig. 1) that varies depending on how much of the peak is captured by the bandpass filter position, it is necessary to correct for this intensity difference prior to image quantification. However, as the silica-encapsulated dye spectra are extremely stable over time, it is only necessary to perform this calibration once for any selected set of SERS peaks.

Fig. 7

(top row) White light (a) and $\mathrm{S}420/421/440/481$ bandpass images (b)-(e) of spatially separated drops of each SERS probe on filter paper, the white circle highlights the laser illumination area. (bottom row) White light and bandpass images of the dorsum of a nude mouse injected with an equimolar mixture of the four SERS reporters. Images were acquired using an integration time of 5 s averaged five times.

To test this multiplex quantification accuracy a triplex mixture of varying amounts of three SERS reporter molecules was imaged in a 96 well plate: one reporter remained constant at 30 pM for all conditions, one increased from (0 to 50) pM, and the third reporter decreased from (50 to 0) pM over six discrete wells. SERS images were taken at all three peak wavelengths and the relative amounts of each were calculated from the known spectral intensities for each reporter. As shown in Fig. 8, the reconstructed amounts of each SERS reporter are in excellent agreement with the actual amounts in each well ($R^2>0.99$). As the cross-channel overlap is generally $<5\%$ for the SERS reporters used here (e.g., S420 has a 4% spectral overlap with S421 and 2% with S481), the overall system sensitivity is not significantly different in the multiplex imaging case than when imaging a single reporter molecule.

Fig. 8

(a) Widefield Raman bandpass image intensities from SERS NP mixtures in a 96 well plate: the concentration of S481 was held constant in all cases while the amount of S421 increased from (0 to 50 pM) in wells 1-6, and the concentration of S420 decreased from (50 to 0 pM) in wells 1-6. (b) SERS image intensities for each mixture as a function of the known concentration of each probe. Error bars represent $\pm 1$ standard deviation of triplicate wells for each case.

In the case of a defined imaging geometry, as in the high-throughput assay plate, the system can be calibrated absolutely against a known dilution series of SERS NPs to quantify the amount of each SERS reporter, allowing for truly quantitative multiplex imaging without the elaborate compensation schemes required in flow cytometry or other fluorescence-based techniques. Leveraging the facile multiplexing in SERS imaging, experimental samples can be spiked with a known quantity of a SERS reporter not used in the assay as an internal standard, thus compensating for any laser intensity variation over the course of an experiment, and so further improving the quantification accuracy.

3.5.

Multiplex SERS Imaging In Vivo

To demonstrate the multiplex imaging capability of this system in vivo, four distinct SERS reporters were immobilized in an acrylamide polymer matrix and implanted subcutaneously into the dorsum of a nude mouse and images were acquired at each of the individual SERS peak wavelengths indicated in Fig. 1. There was no Raman spectral contribution from the matrix material at the measured peak locations (Fig. 9). As shown in Fig. 7, the SERS signal from four distinct reporter molecules were easily detected and separated against the relatively complex background tissue autofluorescence.

Fig. 9

Raman spectra of pure acrylamide, 40 pM bare gold NPs encapsulated in acrylamide, and 40 pM S481 labelled gold NPs encapsulated in acrylamide. There is no spectral contribution of either the acrylamide matrix or the gold core to the Raman bands used for widefield imaging.

As a measure of the quantitative imaging possible with SERS NPs, an additional subcutaneous “cocktail” injection was created with varying amounts of each of the four reporter molecules. As shown in Fig. 10, each probe was detectable above background interference and the measured proportions of each probe based on image intensity are in excellent agreement with the known ratio ($R^2=0.986$). As all the reporter molecules used in this study are structurally related, multiplexing at $4\times$ and beyond does require compensation for cross-talk as many bands begin to overlap within the bandpass of our tunable filter ($4\mathrm{nm}=65{\mathrm{cm}}^{-1}$ at 785 nm excitation)—as an example, S421 has a 10% spectral overlap with S420, 0.1% with S440, and 7.5% with S481.

Fig. 10

White light (a) and $\mathrm{S}420/421/440/481$ bandpass images (b-e) from varying amounts of each reporter molecule injected subcutaneously on the dorsum of a nude mouse. Unmixed Raman bandpass image intensity as a function of the known injected concentration for a quadruplex mixture of SERS reporters in vivo (f). Error bars represent $\pm 1$ standard deviation of three measurements from injections at different locations along the mouse dorsum. Images were acquired using an integration time of 5 s averaged five times.