PHOTOFRIN II UPTAKE BY ATHEROMA IN ATHEROSCLEROTIC RABBITS. FLUORESCENCE AND HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC ANALYSIS ON POST-MORTEM AORTA

Atherosclerotic lesions were induced in normal and Watanabe rabbits by atherogenic diet and stripping of aorta endothelium. The rabbits were injected with Photofrin II and sacrificed two days later. Atheromatous aorta as well as normal aorta from control animals were characterized by their fluorescence spectra using front face excitation. Characteristic emission peaks at 631 and 694 nm were displayed at atheromatous plaques. The excitation spectrum shows a strong band at 394 nm and weaker bands at 446, 504, 536 and 574 nm. Although no fluorescence of normal aorta can be seen by visual inspection, emission with a maximum at 626 nm was detected by spectrofluorimetry. Normal phase high performance liquid chromatography analysis of extracts from atheroma and control aorta were also carried out. The specific labelling of atheroma involves mainly protoporphyrin, hematoporphyrin and also minor components of Photofrin II which are accumulated. Some other components are accumulated but do not appear to be specifically retained by atheroma.     

DISTRIBUTION AND ELIMINATION OF PHOTOFRIN II IN MICE

The distribution and elimination of [14C]PII, the radioisotopically-labeled equivalent of the mixture of porphyrins known as Photofrin II used in the photodynamic treatment of solid tumors, were determined in tumor-free and SMT-F tumor-bearing DBA/2 Ha-DD mice. Following i.p. injection, drug was absorbed from the peritoneum with a half-life of about 1 h; elimination from plasma was rapid, declining about 1.4 logs in concentration over 48 h following i.v. administration. However, some [14C]-activity was still detectable after 75 days. Normal tissues take up the drug within about 7.5 h after administration, with peak concentrations distributed as follows: liver, adrenal gland, urinary bladder greater than pancreas, kidney, spleen greater than stomach, bone, lung, heart greater than muscle much greater than brain. Only skeletal muscle, brain, and skin located contralaterally to subcutaneously implanted SMT-F tumors had peak [14C]-activities lower than tumor tissue; skin overlying SMT-F tumors showed concentrations not significantly different (P greater than 0.3) from tumor. After 75 days all tissues examined retained some fraction of [14C]-activity, ranging from 16% for kidney to 61% for spleen, of the initial peak tissue levels. The primary route of elimination of Photofrin II was through the bile-gut pathway, with greater than 59% of the administered [14C]-activity recovered in the feces, and only about 6% in the urine, over 192 h. HPLC analyses of fecal extracts showed that mostly monomeric and other low molecular weight porphyrin components of Photofrin II were eliminated. The higher molecular weight oligomeric fractions of Photofrin II were retained in liver and spleen up to 14 days after injection.     

IMPROVED SURVIVAL FROM INTRACAVITARY PHOTODYNAMIC THERAPY OF RAT GLIOMA

The effectiveness of intratumoral photoradiation in photodynamic therapy (PDT) using a polyporphyrin photosensitizer was studied in the RT-2 rat glioma model. One week after intracerebral implantation of RT-2 cells, experimental rats received a single i.p. injection of 2 mg/kg of Photofrin. After administration of the photosensitizer (48 h), the tumors were partially resected and the exposed cavity was irradiated with 15 J of laser light at a wavelength of 630 nm. Further treatment with a large craniectomy significantly enhanced rat survival. Control rats which received no photosensitizer but were treated with surgery, alone or in combination with laser irradiation, succumbed from early tumor recurrence. Photodynamic therapy without decompressive surgery resulted in hemorrhagic infarction of residual tumor and adjacent brain with focal cerebral edema which resulted in cerebral herniation and early death. Our results indicate that photodynamic therapy is effective in treating residual brain tumor but at the expense of brain tissue surrounding the tumor. Unless relieved, intracranial pressure from photodynamic therapy-associated cerebral edema in this animal model resulted in shortened survival.     

The influence of photodynamic therapy on the ultrastructure of the normal rat bladder.

Reduced bladder capacity is a major side effect for patients receiving photodynamic therapy (PDT) for bladder cancer. A rat bladder model has been developed to address both the vascular and tissue effects of the photodynamic treatment of the urinary bladder. Bladders were exteriorized and positioned in a plexiglass tissue bath. Effects on microvasculature were assessed during PDT of the bladder by recording luminal diameter changes in arterioles and venules. Animals receiving Photofrin II (10 mg kg-1) 30 min prior to PDT scored a statistically significant reduction in the diameter of the red blood cell column in the vessels, whereas administration of Photofrin II 48 h prior to PDT was ineffective. Morphological changes included significant endothelial and vascular myocyte damage in the 30 min PDT group alone. Among the other tissue components, the mucosal lining was minimally affected and the response of the muscularis was highly variable. Smooth muscle cell changes ranged from mild contraction to frank necrosis with many of the affected cells located near the altered vascular beds. These data suggest that the clinical symptoms of reduced bladder capacity can be accounted for by vascular damage and myocyte sensitivity. Further refinements in the Photofrin II and light doses used in therapy may reduce bladder complications and allow for better management of bladder cancer.     

CHLORIN AND PORPHYRIN DERIVATIVES AS POTENTIAL PHOTOSENSITIZERS IN PHOTODYNAMIC THERAPY

In order to find a photosensitizer with better optical properties and pharmacokinetics than Photofrin II, a series of new photosensitizers related to methyl pheophorbide-a and chlorin-e6 were synthesized. These compounds absorb at substantially longer wavelengths (lambda max 660 nm) than does Photofrin II (630 nm) and show promise for use in photodynamic therapy. Among the porphyrins, we observed that long carbon chain ether derivatives are better photosensitizers than their ester analogs. These sensitizers were tested for in vivo photosensitizing activity vis-a-vis Photofrin II, using the standard screening system of DBA/2 mice bearing transplanted SMT/F tumors. Most of these photosensitizers were found to have better tumoricidal photosensitizing activity than Photofrin II and demonstrated more rapid attenuation of normal tissue photosensitivity with time after administration vis-a-vis Photofrin II.     

Fluorescence Lifetime Spectroscopy of Glioblastoma Multiforme¶

Fluorescence spectroscopy of the endogenous emission of brain tumors has been researched as a potentially important method for the intraoperative localization of brain tumor margins. We investigated the use of time‐resolved, laser‐induced fluorescence spectroscopy for demarcation of primary brain tumors by studying the time‐resolved spectra of gliomas. The fluorescence of human brain samples (glioblastoma multiforme, cortex and white matter: six patients, 23 sites) was induced ex vivo with a pulsed nitrogen laser (337 nm, 3 ns). The time‐resolved spectra were detected in a 360–550 nm wavelength range using a fast digitizer and gated detection. Parameters derived from both the spectral‐ (intensities from narrow spectral bands) and the time domain (average lifetime) measured at 390 and 460 nm were used for tissue characterization. We determined that high‐grade gliomas are characterized by fluorescence lifetimes that varied with the emission wavelength (>3 ns at 390 nm, <1 ns at 460 nm) and their emission is overall longer than that of normal brain tissue. Our study demonstrates that the use of fluorescence lifetime not only improves the specificity of fluorescence measurements but also allows a more robust evaluation of data collected from brain tissue. Combined information from both the spectraland the time domain can enhance the ability of fluorescencebased techniques to diagnose and detect brain tumor margins intraoperatively.     

THE TIME COURSE OF CUTANEOUS PORPHYRIN PHOTOSENSITIZATION IN THE MURINE EAR

This study was designed to investigate the time course of acute cutaneous photosensitivity following administration of Photofrin II using the murine ear swelling response (ESR) as an in vivo end-point. Ros:(ICR) mice were injected with 5 mg/kg Photofrin II and illuminated 7.5 h to 31 days later with 630-nm laser light; ESR was measured 24 h after illumination. There was a direct correlation between ESR and the concentration of [14C]Photofrin II in blood, while no relationship between ESR and the level of [14C]Photofrin II in the ear tissue of exsanguinated mice was evident. Photosensitivity in the mouse foot can be suppressed by preexposure to low doses of light via a photochemical destruction of tissue-bound sensitizer (Boyle and Potter, 1987, Photochem. Photobiol. 46, 997-1001). However, mouse ears pretreated with 84 J/cm2 of 630-nm light (28 J/cm2/day, given 2, 4 and 6 d after injection), a dose sufficient to reduce porphyrin fluorescence in ear tissue by about 75%, prior to the usual light dose (88.6 J/cm2, 630 nm, day 9 after injection) showed a mean ESR not significantly different (P less than 0.5) from that for ears which received only a single dose of 88.6 J/cm2 on day 9. It is concluded, for this animal model, that circulating porphyrin is the source of photoinduced ear-tissue edema and that photobleaching of tissue-bound sensitizer does not attenuate ear-tissue photosensitivity.     

Distribution and pharmacokinetics of Photofrin in human bile duct cancer

Prognosis of patients with bile duct tumors is mostly poor due to late diagnosis and a lack of adequate curative and palliative treatment modalities. To evaluate the potential of photodynamic therapy (PDT) as a novel and alternative treatment approach, we have investigated the uptake and tumor-specific localization of the photosensitizer Photofrin in human biliary tract neoplasms. We have quantified the distribution and the pharmacokinetics of Photofrin in normal and tumor tissue biopsies of the human bile duct by quantitative fluorescence microscopy and digital image analysis of cryosections. Fluorescence intensities (expressed as a percentage of a standard) are 19.0 +/- 11.4% and 25.2 +/- 12.7% for tumors and 10.9 +/- 2.9% and 13.2 +/- 9.1% (mean +/- SD) for normal bile duct tissue at 24 h (n = 5) and 48 h (n = 8) after Photofrin administration (2 mg kg-1 i.v.), respectively, and decrease afterwards in normal bile duct tissue over the period of investigation (4-35 days). The ratios of fluorescence in tumor versus normal tissue are found to be 1.7 +/- 0.7 and 2.3 +/- 1.2 (mean +/- SD) at days one and two after Photofrin administration, respectively. Thus, Photofrin preferentially accumulates in bile duct neoplasms, reaching peak values during the first two days. These data suggest that laser irradiation should be performed within this period after Photofrin injection to achieve tumor selectivity of PDT for effective treatment of bile duct carcinoma.     

Simultaneous two-photon excitation of photofrin in relation to photodynamic therapy

Photodynamic therapy (PDT), the use of light-activated drugs (photosensitizers), is an emerging treatment modality for tumors as well as various nononcologic conditions. Single-photon (1-gamma) PDT is limited by low specificity of the photosensitizer, leading to damage to healthy tissue adjacent to the diseased target tissue. One solution is to use simultaneous two-photon (2-gamma) excitation with ultrafast pulses of near-IR light. Due to the nonlinear interaction mechanism, 2-gamma excitation with a focused beam is localized in three dimensions, allowing treatment volumes on the order of femtoliters. We propose that this will be valuable in PDT of age-related macular degeneration (AMD), which causes blindness due to abnormal choroidal neovasculature and which is currently treated by 1-gamma PDT. Here, Photofrin has been used as the photosensitizer to demonstrate proof-of-principle of 2-gamma killing of vascular endothelial cells in vitro. The 2-gamma absorption properties of Photofrin were investigated in the 750-900 nm excitation wavelength range. It was shown that 2-gamma excitation dominates over 1-gamma excitation above 800 nm. The 2-gamma absorption spectrum of Photofrin in the 800-900 nm excitation wavelength range was measured. The 2-gamma cross section decreased from about 10 GM (1 GM = 10(-50) cm4 s/photon) at 800 nm to 5 GM at 900 nm. Adherent YPEN-1 endothelial cells were then incubated with Photofrin for 24 h and then treated by PDT at 850 nm where the 1-gamma contribution was negligible. Cell death was monitored with the use of 2-gamma scanning laser microscopy. The light doses required for killing were high (6300 J cm(-2) for approximately 50% killing), but 2-gamma cytotoxicity was unequivocally demonstrated. Although Photofrin is, per se, not a good choice for 2-gamma PDT due to its low 2-gamma cross section, this work provides baseline data to guide the development of novel photosensitizers with much higher 2-gamma cross sections (>100 GM), which will be required for 2-gamma PDT of AMD (and other conditions) to be clinically practical.     

Application of charge-coupled device technology for measurement of laser light and fluorescence distribution in tumors for photodynamic therapy

Laser and fluorescence light distributions with applications for photodynamic therapy were measured in mouse tumors using a non-invasive electronic optical imaging system. The system consists of a liquid-nitrogen-cooled, charge-coupled-device (CCD) array camera under computer control with 576 x 384 detection elements having dimensions of 23 microns x 23 microns. The available dynamic range of the array is approx. 10(3), and the effective wavelength range is 400-1000 nm. An interstitially placed cylindrical diffusing optical fiber was used to provide tumor illumination. The light distribution pattern from the fiber was determined by immersing the cylindrical diffusing tip in a fluorescing solution and recording the emission image. Fluorescence imaging facilitates an accurate measurement of light intensity distribution while avoiding problems associated with the directional nature of other detection methods used with diffusing fibers. Radiation-induced fibrosarcoma tumors on C3H mice were grown to about 1 cm diameter for in vivo recording of light distribution from the tumor volume and for determination of effective light penetration distance at 18 wavelengths in the range 458-995 nm. Endogenous tumor fluorescence and Photofrin II fluorescence intensity were measured over the wavelength range 585-725 nm to investigate the possible application of CCD imaging technology for drug distribution measurements. Model experiments were begun to evaluate the relative importance of potential distortions of light distribution measurements using this approach.     

Photodynamic Therapy of 9L Gliosarcoma with Liposome-Delivered Photofrin

Abstract— The effect of Photofrin encapsulated in a liposome delivery vehicle for photodynamic therapy (PDT) of the 9L gliosarcoma and normal rat brain was tested. We hypothesized that the liposome vehicle enhances therapeutic efficacy, possibly by increasing tumor tissue concentration of Photofrin. Male Fisher rats bearing a 9L gliosarcoma were treated 16 days after intracerebral tumor implantation with either Photofrin in dextrose (n = 5) or Photofrin in liposome (n = 6). Nontumor-bearing animals were treated with Photofrin delivered either in dextrose (n = 4) or liposome (n = 4) vehicle. Tissue concentrations of Photofrin delivered either in dextrose (n = 4) or liposome (n = 4) vehicle were measured in tumor, brain adjacent to tumor and in normal brain tissue. Photofrin was administered (intraperitoneally) at a dose of 12.5 mg/kg and PDT (17 J/cm₂ of 632 nm light at 100 mW/cm₂) was performed 24 h after Photofrin administration. Brains were removed 24 h after PDT and stained with hematoxylin and eosin for analysis of cellular damage. The PDT using Photofrin in the liposome vehicle caused significantly more damage to the tumor ( P < 0.001) than did PDT with Photofrin in dextrose. The PDT of tumor with Photofrin delivered in liposomes caused a 22% volume of cellular necrosis, while PDT of tumor with Photofrin delivered in dextrose caused only scattered cellular damage. Photofrin concentration in tumors was significantly higher ( P = 0.021) using liposome (33.8 ± 18.9 μg/g) compared to dextrose delivery (5.5 ± 1.5 μg/g). Normal brain was affected similarly in both groups, with only scattered cellular necrosis. Our data suggest that the liposome vehicle enhances the therapeutic efficacy of PDT treatment of 9L tumors.     

PHOTODYNAMIC THERAPY-INDUCED HYPOXIA IN RAT TUMORS and NORMAL TISSUES

Abstract The administration of misonidazole (MISO) to Fischer x Copenhagen rats whose R3327-H prostate tumors were treated with photodynamic therapy (PDT) produced enhanced tumor growth delays and cures. This potentiation of PDT by MISO was previously observed with R3327-AT tumors and was postulated to result from drug cytotoxicity of naturally-occurring and PDT-induced hypoxic cells. Radioactively-labelled MISO has been developed as a marker for tissue p0₂ at the cellular level and [³H]MISO was administered to R3327-AT and R3327-H tumor-bearing rats before and after standard PDT treatments. The amount of ³H in tissues 24 h after drug administration was a measure of'bound MISO'which reflects average tissue oxygenation. [³H]MISO retained in R3327-AT tumors was ˜4x and in liver tissue ˜2x that retained in muscle, heart, brain and R3327-H tumors (1x). Tumors treated with Photofrin II and lased with 1000 J showed a 6-fold increase in retained [³H]MISO in R3327-H tumors and a 2-fold increase in retained [³H]MISO in R3327-AT tumors. The absolute levels of retained ³H in both tumors after PDT were similar. These data provide direct evidence that PDT induces rapid hypoxia in both tumors. When the gastrocnemius muscle of the rat leg was similarly treated, the amount of [³H]MISO retained was ˜4x greater than that in untreated muscle. This result suggests that PDT-induced hypoxia is not selective to just tumor tissue. These data suggest that the hypoxia-inducing property of PDT might be exploited in combination with hypoxic cell cytotoxins to produce improved tumor responses and cures.     

Biodistribution of Photofrin II® and 5-Aminolevulinic Acid-Induced Protoporphyrin IX in Normal Rat Bladder and Bladder Tumor Models: Implications for Photodynamic Therapy

Photodynamic therapy (PDT) has been considered as a potential therapy for superficial bladder carcinomas. Cutaneous photosensitivity and reduction of bladder capacity are the two well-known complications following systemic administration of the commonly used photosensitizer, Photofrin II® (PII). The objective of the present study was to evaluate whether intravesical. (i.b.) instillation of photosensitizers for PDT of bladder cancer might be a more suitable treatment method. Female Fischer rats were utilized to develop orthotopic and heterotopic bladder tumor models. Rats bearing orthotopic bladder tumors were treated either intravesically or intravenously with graded doses of 5-aminolevulinic acid (ALA) or PII. Normal rats received the same doses of ALA or PII. As well, rats bearing heterotopic tumor were studied for comparison. The biodistribution times (times allowed for tissue uptake and bioconversion following drug administration) were 2, 4 or 6 h. Porphyrin fluorescence intensities within tumor, urothelium, submucosa, bladder muscularis and abdominal muscle were quantitated by confocal laser scanning microscopy. Following intravenous (i.v.) injection of ALA, tumor protoporphyrin IX (PpIX) levels peaked at 4 h and diminished by 6 h. The PpIX ratios of tumor-to-bladder mucosa, submucosa and muscle layers were 3:1, 5:1 and 8:1, respectively, 4 h following 1000 mg/kg ALA injection. After ALA instillation, the optimal biodistribution time appeared to be 4 h. Bladder instillation provided comparable tumor labeling with the i.v. route, but lost selectivity of PpIX accumulation between tumor and normal urothelium. The PpIX ratio of tumor-to-bladder muscularis was 5:1. After i.b. instillation of PII, porphyrin fluorescence was detected only within tumor and urothelium, while porphyrin fluorescence was mainly located in bladder submucosa following i.v. injection. Intravesical administration of ALA or PII might be feasible for PDT of superficial bladder cancers.     

INTRAUTERINE 5-AMINOLEVULINIC ACID INDUCES SELECTIVE FLUORESCENCE AND PHOTODYNAMIC ABLATION OF THE RAT ENDOMETRIUM*

Abstract— 5-Aminolevulinic acid (ALA), a precursor of protoporphyrin IX (Pp IX), was administered into the rat uterine cavity in an attempt to selectively ablate the endometrium. Doses of ALA ranging from 4 to 50 mg were injected into one uterine horn of rats while the vehicle (saline) was injected into the contralateral horn. Animals were divided into three groups. In group one, the uterine horns were removed and processed for either fluorescent mi- croscopy or spectrophotofluorometry 3 h later. In group two, rats were allowed to survive for either 2 or 10 days, and then the uterine horns were harvested and processed histologically. In group three, both uterine horns were exposed to transmural light (approximately 150 J/cm*) 3 h after administration of ALA or saline and processed histologically either 2 or 10 days later. Fluorescent microscopy showed fluorescence in the endometrium and not in the myometrium. The maximum emission spectra of endometrial fluorescence occurred at 630 and 690 nm, characteristic of Pp IX. In contrast, no fluorescence was detected in saline-treated uterine horns. Light exposure resulted in extensive damage only to the ALA-treated endometrium. There was no indication of regeneration 10 days after treatment. We conclude from these studies that ALA administered into the lumen of the rat uterus is selectively converted into Pp IX within the endometrium. Furthermore, photoactivation of the Pp IX results in selective ablation of the endometrium.     

Time-resolved fluorescence spectra of arterial fluorescent compounds: reconstruction with the Laguerre expansion technique

The time-resolved fluorescence spectra of the main arterial fluorescent compounds were retrieved using a new algorithm based on the Laguerre expansion of kernels technique. Samples of elastin, collagen and cholesterol were excited with a pulsed nitrogen laser and the emission was measured at 29 discrete wavelengths between 370 and 510 nm. The expansion of the fluorescence impulse response function on the Laguerre basis of functions was optimized to reproduce the observed fluorescence emission. Collagen lifetime (5.3 ns at 390 nm) was substantially larger than that of elastin (2.3 ns) and cholesterol (1.3 ns). Two decay components were identified in the emission decay of the compounds. For collagen, the decay components were markedly wavelength dependent and hydration dependent such that the emission decay became shorter at higher emission wavelengths and with hydration. The decay characteristics of elastin and cholesterol were relatively unchanged with wavelength and with hydration. The observed variations in the time-resolved spectra of elastin, collagen and cholesterol were consistent with the existence of several fluorophores with different emission characteristics. Because the compounds are present in different proportions in healthy and atherosclerotic arterial walls, characteristic differences in their time-resolved emission spectra could be exploited to assess optically the severity of atherosclerotic lesions.     

LASER-INDUCED FLUORESCENCE IN MALIGNANT and NORMAL TISSUE OF RATS INJECTED WITH BENZOPORPHYRIN DERIVATIVE

Abstract— Laser-induced fluorescence was used to characterize the localization of intravenously administered ben-zoporphyrin derivative-monoacid (BPD-MA) 3 h postinjection in different rat tissue types, including an induced experimental malignant tumor. A comparison of the fluorescence properties and the demarcation potential between the newer sensitizer BPD-MA and four other substances, hematoporphyrin (HP), polyhematoporphyrin ester (PHE), tetrasulfonated phthalocyanine (TSPc) and the commercially available Photofrin earlier investigated, is included. The fluorescence light was induced with a nitrogen laser, emitting at 337 nm. The fluorescence spectrum in the region380–750 nm was analyzed by a polychromator equipped with a diode array detector. The demarcation potential between tumor and surrounding tissue in terms of fluorescence signal for the tumor model used was 2:1 for BPD-MA. In comparison with the other drugs, HP shows about the same demarcation potential, whereas Photofrin and PHE exhibit about 3 times better and TSPc about 1.5 times better demarcation. By also employing the endogenous tissue fluorescence signature the contrast was enhanced by a factor of about 2 for each of the five drugs.     

In vivo Laser-induced Fluorescence Imaging of a Rat Pancreatic Cancer with Pheophorbide-a

Laser-induced fluorescence (LIF) of pheophorbide-a (Ph-a) was used for imaging of a rat pancreatic tumor. Using a dimensionless function (the ratio of Ph-a fluorescence by bluish autofluorescence), the fluorescence contrasts between excised tumors and their paired pancreas were investigated up to 48 h after a 9 mg kg^-1 Ph-a intravenous administration. Among five tested excitation wavelengths, 355 and 610 nm excitations gave the best distinctive contrasts, both 48 h after dye injection. The LIF imaging of six intrapancreatic tumors and six healthy pancreas was carried out in vivo using two laser excitations: 355 nm (Nd:YAG + tripling) for bluish autofluorescence and 610 nm (rhodamine 6G dye) for reddish autofluorescence and dye emission. Images were recorded through bandpass filters at 470 and 640 nm (autofluorescence) and at 680 nm (dye + autofluorescence) with an intensified charged-coupled device camera. Autofluorescence as Ph-a fluorescence images did not allow accurate LIF diagnosis of pancreatic carcinoma. An image processing, including for each pixel a computed division of Ph-a fluorescence (after subtraction of reddish autofluorescence) by bluish autofluorescence intensity generated poorly contrasted tumor images in five of six and false tumor localization in one of three of the tumor-bearing pancreas. A fitting of the digital 640 nm autofluorescence up to the mean 680 nm fluorescence intensity in pancreas prior to subtraction allowed a safe diagnosis to be made with well-contrasted tumor images. To assess automation ability of the processing, a same fitting coefficient (mean of individual values) was applied. In this way, false-negative (one of six) and false-positive (two of six) images were present in tumor-bearing animals as false-positive in one-half of the controls. A successful standardized procedure was then applied with a normalization of 640 and 680 nm pancreas intensities to a same set threshold prior processing. In opposition to thin-layered hollow organs, such as bronchial tube or digestive tract, LIF imaging of carcinoma inserted in a compact organ is exhausting. The use of a dye excitable in the red wavelength range (610 nm for Ph-a) may partly solve this problem, rendering LIF imaging more accurate and potentially automated.     

RATIOING FLUOROMETER PROBE FOR LOCALIZING CARCINOMA IN SITU IN BRONCHI OF THE LUNG

Abstract A bronchoscopic ratioing fiuorometer probe, that ratios the red light emitted from bronchial tumor to the green light of autofluorescence upon stimulation by violet light (413 nm) has proven capable of detecting and localizing small bronchial tumors bronchoscopically, 4, 48, and 72 h after intravenous Photofrin II injection. Still to be determined is whether'field averaging'in the case of very small bronchial carcinoma in situ will diminish accuracy. The accuracy, efficiency, and cost of localization by imaging fluorescence bronchoscopy will be compared to that by non-imaging ratioing fiuorometer probe.     

Fluorescence spectrometry for quantitative characterization of cobalt(II) complexation by Leonardite humic acid

Quenching of the fluorescence of a Leonardite humic acid by Co(II) has been studied at different pH. The interaction was monitored by emission fluorescence and by synchronous fluorescence with two different offsets (deltalambda=20 and 80 nm). It was found that synchronous fluorescence performed with the smaller offset resolves the individual components of the heterogeneous material better than emission or synchronous fluorescence performed with the larger offset. Enhancement of the signal induced by Cobalt(II) complexation resulted in more complex behavior for measurements performed by synchronous fluorescence with an offset of 20 nm, however. The quenching profiles obtained for pH 5.0, 6.0, and 7.0 ([KNO(3)]=0.1 mol L(-1); [LHA]=3.3 mg(C) L(-1); [Co(II)]=1.0 x 10(-6)-1.6 x 10 (-3) mol L(-1)) by emission and synchronous (deltalambda=80 nm) fluorescence were analyzed by two methods: 1. a non-linear least-squares procedure that leads to conditional constants; and 2. a pH-dependent discrete logK spectrum model that leads to stability constants. The first method resulted in poor fitting and unreasonable values for maximum capacities. The second procedure resulted in smooth fitting that accounted well for the pH changes when results for pH 6.0 and 5.0 were predicted by use of the four values of logK(Co)(i) (4.31, 3.76, 7.32, and 7.67 corresponding to the four sites (i) of the respective pKa(i) values 4, 6, 8, and 10) calculated at pH 7.0 for the equilibrium     

QUANTITATIVE in vivo MEASUREMENT OF THE FLUORESCENT COMPONENTS OF PHOTOFRIN II

Abstract Photofrin II which contains the most efficient components of hematoporphyrin derivative with regard to photodynamic therapy of cancer, was measured fluorometrically in tumor and tumor-free tissues in vivo over a period up to 8 days. Using time-resolving (nanosecond and picosecond) microscopic techniques, the fluorescence of different components was quantitated and attributed to monomelic, dimeric, and possibly aggregated porphyrin species. The long-lasting retention of the porphyrins in live tissues was in contrast to the rapid removal from cultured cells. This might be due to monomerization or dimerization of non-fluorescent aggregates. Tumor-selective accumulation was found to be similar for two different (probably monomeric and dimeric) components. This indicates that the integral fluorescence of these components may also correlate with the distribution of the main photosensitizing species.