Similarly figure 4-8 shows fluorescence from the same phantoms when m onitored using
the corresponding experimental setup (described in Chapter 3), with excitation at 632nm
from a low pow er H eN e laser, with the fluorescence emissions normalised to the scattered excitation light detected at 632nm. The intensity o f the fluorescence emission
depends on the concentration o f the PS contained in the sample, with the peak at 680nm
increasing with increasing photosensitiser concentration
0.5 H ^ 0.4 - U) c
I
. > 0.2 4 0.3 - TO (D OC 0 .0 -I 630 640 650 660 670 680 690 700 Wavelength /nm 0.25 -1^
0.20 - Ü) I 0.15 - 0.10 - 0.05 -B
<u > §Si
0.00 ■ -O @ 670nm @ 685nm ... □ .-a-" ..-o - . . . D - ... ...o ... -o h O 'O ' 0.0 0.5 1.0 1.5 2.0 Concentration /|aM 2.5 3.0Figure 4-8 Fluorescence spectra front phantoms with different concentrations o f photosensitiser and the intensity o f the recorded signal at 670 and 68Snm,
Reflectance & Fluorescence 90
It must be noted that at higher concentrations o f photosensitiser the fluorescence emission undergoes a slight red-shifl o f about lOnm, which is due to the self absorption. The absorption bands centred at 670 and 610nm represent the fluorescent monomer which is shown in figure 4-8 A. Owing to scattering o f the light the effective pathlength is longer than the 3mm fibre separation, so a low concentration o f photosensitiser results in a larger absorption than it would in a clear solution with similar detection geometry. From the data presented above it is clear to see that fluorescence emission from the photosensitiser is easier to monitor at low concentrations than the diffuse reflectance spectra, since with absorbance w e are looking for a small change in a large signal and with fluorescence you may record a small signal in a very low background (if noisy). It is always easier to detect the presence o f something rather than the absence. Figure 4-8 B demonstrates that it is possible to detect the fluorescence wavelength shift by monitoring the intensity at specific wavelengths, such as could be carried out with a photodetector (e.g. photomultiplier tube, PMT) and filter bandpass allowing detection o f fluorescence) detection arrangement. This problem does not occur when using a CCD/spectrograph system since the entire spectral range is recorded.
At higher concentrations o f the AlCIPc an absorption band at approximately 635nm is clearly apparent on the diffuse reflectance spectra (figure 4-6 & 4-7). This displays one o f the advantages o f using Rd to monitor chromophore concentration since not all the light absorbed is emitted as fluorescence - the dimer o f AlCIPc does not fluoresce but it clearly absorbs as seen in figure 4-7. The absorption noted on the graph is due to dimerization o f AlCIPc, the dimer absorbs light at ~635nm but does not fluoresce, therefore the presence o f the dimer is not apparent in the fluorescence spectra. Aggregation has been shown to be evident with this photosensitiser in liposomes (Dhami & Phillips, 1996), with less dimérisation occurring with lower photosensitiser concentrations. (One must remember however that the excitation source for fluorescence monitoring can be set to a wavelength where the monomer will absorb the light but the absorption fi’om the dimer is negligible). The effect o f dimerization o f phthalocyanines may be important during PDT and will be discussed in detail with regard to photobleaching in Chapter 5.
Increasing the concentration o f photosensitiser in the sample affects the absorption o f the excitation light as discussed in section 4.2.3. The higher the concentration then the less o f the light passes through the sample resulting in fluorescence. It is this effect, known as self shielding, which has been shown to be important with the new second generation photosensitisers which have high absorption coefficients. It is shown in figure 4-9 that the difference in the intensity o f the scattered light varies with concentration, in this case AlCIPc. Unfortunately the difference is not very pronounced and therefore this method o f monitoring concentration changes will not be used on living tissues.
1 0. 1 0 . 0 1 1 0 6 8 0 2 4 F i b r e s e p a r a t i o n / m m
Figure 4-9 Changes in the detected excitation light with concentration and fibre separation - note log scale.
Reflectance & Fluorescence 92
4.4.4 Effect of pathlength on LIE and Rj
The separation between the excitation fibre and the detection fibre determine the optical pathlength through the tissue. Increasing the separation increases the volume o f the tissue being sampled (size o f the banana shape through the tissue, Stratonnikov et al, 1996) and the number o f absorption/scattering interactions that the average photon encounters before detection also increases. The relative changes in the fluorescence signal and the reflectance signal therefore depend on the absorption coefficient and the scattering coefficient o f the sample (section 2.2.2.2). A s the pathlength is increased, the probability that a photon will reach the detector without being absorbed is reduced, which results in an increase in the relative absorption o f the excitation light between the source and detector fibres. The increase in the absorption manifests itself in a relative increase in the dip in the reflectance spectrum compared to a control. The entire spectrum is a lot less intense since less photons will be scattered in the direction o f the detector fibre as the distance is increased. In effect, the volume that is being sampled is increased and the absorption o f the light is averaged over this larger volume. In order to normalise the reflectance spectra to show the clear difference in the absorption dips a wavelength is selected where the photosensitiser (AlCIPc) does not absorb (Dhami & Phillips, 1996) and the spectra are normalised to this wavelength (740nm). The results o f such normalisation are shown in figure 4-10.