Figure 7: (a) CARS spectrum of pure PS. (b) CARS spectrum of pure PMMA. (c) Image constructed by taking the spectrum at each point across a spin-cast mixture of PS and PMMA. The image contrast is based on the 1000/800 cm–1 peak ratio. Note that a ratio of 0.5 corresponds to the PMMA spectrum (blue) and a ratio of 4 signifies that mostly PS is present (red). Here, one can also see the relative amounts of both components in the regions where they are mixed.

Figure 7 shows the results for the PS–PMMA spin-cast film sample. Again, the spectra for the individual components are shown (Figures 7a and 7b). However, because the spectra for the two polymers have similar features around both 1000 cm^–1 and 800 cm^–1, we analyzed the ratio of the these peak intensities for each pixel in the image. The contrast scale for the image in Figure 7c is shown. It is a false-color image in which the bright red corresponds to 1000 cm^–1 /800 cm^–1 = 4, which is the ratio of the spectral peaks in the pure PS spectrum, and the dark blue is 1000 cm^–1 /800 cm^–1 = 0.5, corresponding to pure PMMA.

The spatial resolution in Figure 6 and Figure 7 is about 400 nm, which is less than ideal. For 800-nm light, the best spatial resolution observed to date is 230 nm due to the high numerical aperture of the focusing objective (1.2 NA) and the third-order nonlinearity of the CARS signal, which gives an enhancement of the spatial resolution over a one-photon process (27). The degradation of the spatial resolution is most likely the result of chromatic and achromatic aberrations and misalignment through the focusing objective, which can be significant due to the exceptionally broad bandwidth of the spectrum. Due to the increased demand for near-IR applications in microscopy, newer microscope objectives have improved aberration compensation capabilities in the region of interest to us (28). This should allow for significant improvements in the future.

In addition to imaging multiple species simultaneously, there are two main advantages to taking a spectrum at each pixel and doing a peak ratio analysis for chemical contrast. First, as seen with the PS and PMMA sample, in mixed samples there are often overlapping peaks in the spectra of the molecules of interest. By looking at peaks individually, such as in the first example of PDMS and DMF, true chemical selectivity cannot be achieved for samples whose components have the same or similar spectral features. Second, components in a sample are not always separated into spatially distinct domains, but often are mixed. Using a peak ratio contrast mechanism gives information about the relative proportions of all components simultaneously at each pixel in the image. For example, in Figure 7c, the red regions are pure PS, and the dark blue regions are pure PMMA, but the light yellow and light blue regions in this figure correspond to mixtures of the two polymers. It is evident that the polymers separate into domains tens to hundreds of micrometers in size, but with smaller islands of pure and mixed composition interspersed within these larger domains. AFM images of these spin-cast PS–PMMA films (not shown) indicate that separate regions also differ in thickness from 50 nm to 700 nm, depending upon the initial concentrations of the polymers in solution. For more complex samples, one can utilize the entire spectrum rather than fitting just two distinct peaks, as was done here.