In the course of experiments for our FET open project Lumiblast, we set off to measure the excitation
of various photoactive drugs (photosensitizers, PS) by the luminescence emission of luminol. Luminol
(5-Amino-2,3-dihydrophthalazine-1,4-dione) is a chemical that interacts with reactive oxygen species
(ROS) in basic conditions, and in the presence of metal catalysts like Fe or Cu, gives out a characteristic
blue luminescence. When dissolved in organic solvents like DMSO, however, luminol only requires
the addition of bases like KOH, NaOH or potassium terbutoxide, to fulfil the conditions for
luminescence emission.
In the present work we employed a detection system based on a spectrograph coupled to a ccd camera
to register fluorescence (Fig 1B) or luminescence (Fig 1 A, C). In the case of characteristic fluorescence
registration (Fig. 1B), the PSs investigated were excited by a 532 nm laser with a variable power output.
We have documented the energy transfer from chemically induced luminol luminescence to a number
of PSs including rose bengal, erythrosin B, hypericin amongst others. In all cases both the luminol
emission and the luminol luminescence-induced PS fluorescence were registered as shown in the
example of luminol and erythrosine b in Fig. 1C.
We further attempted to register the generation of singlet oxygen from luminol-excited PSs. To achieve
this, we employed the near-infrared (NIR) photomultiplier tube (PMT) shown in Fig.1 E, with a cut-off
filter at 900nm and a bandpass filter at 1270±30 nm. This allowed only radiation within this spectral
region to reach the PMT, corresponding to the characteristic phosphorescence of singlet oxygen, spin
forbidden de-excitation to ground state triplet oxygen.
A characteristic steady state singlet oxygen registration can be seen in Fig. 1D, for erythrosine b which
has a high singlet oxygen quantum yield. The luminol luminescence was initiated by addition of
terbutoxide to the DMSO luminol solution, at which point we can see a rise of the signal at 1270 nm.
Upon addition of the singlet oxygen quencher, L-histidine, the signal dropped steeply to background
levels.
NOTE: Figures are not available.
A novel detection method for malignant tumour detection through specific accumulation of far-red emitting photosensitisers, has been developed. The set-up, based on He-Ne (633 nm) excitation was tested with phantom malignancies models using photosensitisers on chicken tissue. Intensity vs. depth of tissue measurements as well as photobleaching measurements were performed. Point by point fluorescence imaging indicated that the formation size is not significantly distorted with respect to depth of location.
The B 2(Sigma) +->X2(Sigma) + violet band emission spectrum of CN following laser ablation of type I collagen was exploited for the estimation of the transient temperature of the plasma plume and consequently of the sample surface considering thermodynamic equilibrium. The temperature dependence upon experimental parameters, such as laser intensity and wavelength as well as delay of gated detection was obtained. The temperature was found to increase with increasing laser intensity and decrease with increasing gated time delay and laser wavelength.
Under pulsed nanosecond laser irradiation of type I pure collagen over the 760 - 1070 nm spectral range, optical up- conversion of the incident radiation is observed. More specifically irradiation of collagen at 1064, 901, 892, 828, 785 and 766 nm produces monochromatic second harmonic signals at half the original wavelengths i.e. 532, 451, 446, 414, 393 and 383 nm respectively. The dependence of the second harmonic signal on the excitation intensity was found to be quadratic [log(I532) equals 1.92*log(I1064]. A weaker third harmonic signal was also observed from collagen at 355 nm when irradiated by 1064 nm nanosecond pulses. This signal was found to bear a near cubic dependence upon the irradiation intensity ]log(I356) equals 2.53*log(I1064)]. The polar distribution of the second harmonic radiation was recorded for both pure fibrous collagen as well a for collagen diluted in acetic acid and forming a semitransparent dry film. In the latter case, significant optical behavior was demonstrated, potentially important for further studies of these nonlinear phenomena and for novel applications.
The photoinduced effects of hypericin, a polycyclic quinone, on collagen has been investigated. It was found that after laser irradiation at both 532 nm and 337 nm, the spectral form of triple helix structure collagen fluorescence, changed to a spectral profile bearing resemblance to that of its polypeptide single chain counterpart, gelatin, or heated collagen. The effect of Chlorin e6 on collagen was also investigated and proved to be dissimilar to that of hypericin and not indicative of profound structural alterations. Second Harmonic Generation (SHG) of 1064 nm- nanosecond laser radiation in collagen was studied. While it was very efficient for pure collagen, the signal intensity was found to diminish by at least an order of magnitude after hypericin photosensitization or heating. The above noted fluorescence spectra form alteration was also observed in a smaller scale in collagen rich chicken tissue (tendon). Non sensitized chicken tendon tissue exhibited very efficient SHG, unlike skin and artery samples.
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