Most of the work presented in this section is published in [3] and [5] (Appendix A.3 and A.5, respectively).
The setup, see section 5.1.3, offers the possibility to perform sPA with excitation light bands situated between 500 nm and 840 nm. To show the usefulness of sPA three different samples were prepared, based on Rhodamine B, Malachite Green and ICG. These are used since their absorption spectra cover three different regions within our accessible wavelength range. The three dyes were each mixed with sil- icone (3140 RTV Coating, Dow Corning). Silicone was chosen because it is fairly easy to obtain and will solidify by letting it to dry for 24 hours, as recommended by Dow Corning. The dyes will then be trapped in the silicone, water can be placed on top of the sample to allow acoustic coupling without destroying the sample. Fig- ure 5.20 represents the absorption spectra of the dyes adapted from literature. Each value corresponds to an integration of the absorption spectra over a 50 nm band- width centred at the corresponding wavelength. Each curve is then normalised over its maximum value. The absorption spectra depend on the dilution medium
FIGURE 5.20: Absorption spectra of Rhodamine B, Malachite Green and ICG, adapted from literature [166, 171, 172].
(a) Rhodamine B
(b) Malachite Green
(c) ICG
FIGURE5.21: Photos and microscope images of the three silicone-dye samples: (a) Rhodamine B, (b) Malachite Green and, (c) ICG.
Scale bars: 5 mm and 0.5 mm (left and right, respectively).
used for the dye. Unfortunately, no literature references are available for silicone. The literature curves presented in this section are taken for Rhodamine B diluted in ethanol, Malachite Green in water and ICG in water with a concentration of 65 µM (absorption spectrum concentration-dependant) [166, 171, 172]. Figure 5.21 shows photos and microscope images of the three silicone-dye samples. Fluctuations in dye concentration can be observed by looking at the changes in color (darker = more concentrated).
Spectroscopic PA measurements were performed over the 500-840 nm range with central wavelengths taken every 25 nm from 500 nm to 800 nm. The influence
(a) Rhodamine B
(b) Malachite Green
(c) ICG
FIGURE5.22: sPA measurements for different bands of (a) Rhodamine B, (b) Malachite Green and, (c) ICG in comparison to their absorption spectra
of the bandwidth of the excitation bands was studied. Three different bandwidths were used: 15 nm, 25 nm and 50 nm. Narrower bandwidths could not be realisti- cally used since less than 10-15 nJ per band is measured on sample for a bandwidth of 10 nm, see Fig. 5.12c. The measurements with a bandwidth of 50 nm were per- formed on the three silicone-dye samples. However, for narrower bandwidths, no measurements were performed on Rhodamine B since too little absorption was ob- tained with the low energy available at shorter wavelengths. Figure 5.22 shows the sPA measurements. The bandwidth used is specified in the legend of each figure. Since the illumination band is changed, it is important to compare with the absorp- tion spectra integrated over the corresponding bandwidth. Very similar curves are obtained regadless of the excitation bandwidth. The error on each PA signal am- plitude measured is ∼ 20 %. In addition, by performing several measurements, the position of the sample may have changed slightly. We can conclude that the bandwidth of the excitation has no influence on the reconstruction of the spectra. However, it can be observed that the absorption spectra obtained with sPA are broader than the one adapted from literature, measured by transmission. We con- jecture that the silicone may influence the sample absorption spectrum. By itself, silicone does not absorb, however it will influence the chemical structure of the dye. It can be observed in Fig.5.21 that the dyes are not uniformly mixed and form aggregates. Aggregates have different properties than uniformly dissolved materi- als [173]. Therefore, aggregates may be responsible for this broader absorption of the dye-samples. The measurement of the absorption spectra by transmission of the samples would permit to confirm these speculations or help us understand this broadening. Thermal effects may influence the sPA measurements as well since several pulses are sent to the sample at the same position (no scanning), heat may occur.
ICG absorption spectra depend on their concentration, the literature curves of Fig. 5.23 represent the ICG absorption spectra corresponding to three different ICG concentrations in water: 6.5 µM, 65 µM and 650 µM. Since our sample does not present a homogeneous concentration, see Fig. 5.21c, sPA was performed at three different positions on the sample. Different concentrations (C1, C2 and C3) lead to fluctuations in the absorption spectra. At low concentrations, the maximum of absorption (e.g. 6.5 µM) is situated at ∼ 780 nm and at ∼ 695-700 nm at higher concentrations (e.g. 650 µM). For intermediate concentrations (e.g. 65 µM), the ab- sorption spectra present two maxima. By looking at the shape of the sPA curves of Fig. 5.23, corresponding to three different positions on the sample, we can conclude that: C1 < C2 < C3 < 650 µM.
FIGURE5.23: sPA measurements of ICG in comparison to ICG absorption spectra taken from literature [172] for different concentrations.
The above experiments demonstrate that sPA can successfully recover the ab- sorption spectra of three silicone-dye samples over a wide wavelength range ex- tending from 500 nm to above 800 nm. The bandwidth of the excitation light has shown very little influence on the spectra obtained. However, less energy is acces- sible on the sample by using smaller bandwidths which may be critical in terms of signal-to-noise ratio and when aiming to perform MPAM on biological tissue over few millimetres laterally.