Biomedical subjects

B J Tromberg

Publications and source records attributed to B J Tromberg.

At least 37 records · Page 2Linked to original sources

PubMed1999

Intrauterine light probe for photodynamic ablation therapy.

BACKGROUND: Endometrial ablation by means of photodynamic therapy is currently being evaluated as an outpatient treatment for dysfunctional uterine bleeding. Photodynamic therapy requires the activation of a photosensitizer by laser light. We describe a new device specifically designed to provide light delivery to the uterus for endometrial photodynamic therapy. INSTRUMENT: The intrauterine light probe consists of the three flexible optical fibers converging to one bundle resembling the shape of the uterine cavity. Each of the fibers contains a cylindrical light diffuser. EXPERIENCE: The intrauterine light probe was tested in removed human uteri for its capability to distribute light in a tissue-simulating scattering medium and to deliver sufficient light throughout the endometrium. The light distribution of the intrauterine light probe in the scattering medium is uniform on eight axes tested around the diffusing fibers. The pattern of light distribution in human uteri is similar to that in the medium. At the endomyometrial junction, there is still one third of the light applied to the endometrial surface whereas deeper in the myometrium, the light power drops to less than 10%. CONCLUSION: We propose a device that will deliver light to the uterine cavity to induce endometrial ablation by means of photodynamic therapy.

Endometrium↗

PubMed1998

Laser-micropipet combination for single-cell analysis.

Due to its potential for exquisite mass detection limits and resolving power, capillary electrophoresis is used for biochemical measurements on single cells; however, accurate measurements of many physiological parameters require sampling strategies that are considerably faster than those presently available. We have developed a laser-based technique to lyse single, adherent, mammalian cells on millisecond time scales. The cellular contents are then introduced into a capillary where electrophoretic separation and detection are performed. Improved temporal resolution of biological measurements results from the extremely rapid lysis made possible by this method. Additionally, the cell is not perturbed by mechanical or electrical stresses prior to sampling. Such disturbances can alter cellular physiology, resulting in inaccurate measurements. The fast cell lysis, the absence of cellular stresses prior to lysis, and the application to adherent mammalian cells are significant refinements to CE-based measurements on single cells. With this laser-micropipet combination, it will be possible to measure the intracellular concentration of molecules that change on subsecond to second time scales, for example, substrates of many cellular enzymes.

Animals↗

PubMed1998

Uptake of the photosensitizer benzoporphyrin derivative in human endometrium after topical application in vivo.

STUDY OBJECTIVE: To determine both the time leading to maximum endometrial drug uptake and distribution of the photosensitizer benzoporphyrin derivative-monoacid ring A (BPD-MA) after intrauterine instillation (Canadian Task Force classification ). DESIGN: Assessment of histology specimens (Canadian Task Force classification I). SETTING: University-based facility. PATIENTS: Twenty-two women scheduled for hysterectomy. INTERVENTIONS: We instilled 1.5 ml of a 2 mg/ml of BPD-MA-Hyskon solution into the uterine cavity of 22 women before hysterectomy. The fluorescence induced was measured by fluorescence microscopy on frozen sections of uterine samples from 20 of 22 patients. Systemic uptake of BPD-MA was determined in plasma of six patients by spectrofluorometry. MEASUREMENTS AND MAIN RESULTS: The BPD-MA-induced fluorescence was maximum 1 hour after instillation, with significantly higher uptake in endometrial glands than in underlying stroma. Hormonal endometrial stimulation correlated with fluorescence intensity: atrophy < secretory phase < proliferative phase. Strongest fluorescence was seen in endometrial cancer. Drug uptake by endometrial glands was found at a depth of 2 mm from the surface. Systemic uptake of BPD-MA was under the detection level of 2 ng/ml after application. CONCLUSION: Fluorescence in human endometrial glands suggests that selective destruction of human endometrium with photodynamic therapy may be possible 1 hour after topical application of BPD-MA for benign and malignant lesions. No systemic drug uptake, side effects, or major technical difficulties were detected. Limited penetration of the drug and selective uptake by endometrial glands provided a high degree of safety for endometrial ablation.

Administration, Topical↗

PubMed1998

In vivo detection of metastatic ovarian cancer by means of 5-aminolevulinic acid-induced fluorescence in a rat model.

STUDY OBJECTIVES: To determine the feasibility of macroscopic visualization of small ovarian cancer metastases in vivo by fluorescence after intravenous administration of 5-aminolevulinic acid (ALA); to assess the time after drug injection when fluorescence of small metastases is maximum; and to correlate macroscopic in vivo fluorescence with both microscopic ex vivo fluorescence and histologic findings. DESIGN: Controlled animal study (Canadian Task Force classification I). SETTING: University-based facility. SUBJECTS: Twenty-four healthy, female Fischer rats. INTERVENTION: Diffuse peritoneal metastatic cancer was induced in Fischer 344 rats by intraperitoneal injection of 1 million syngeneic ovarian cancer cells (NuTu-19). Four weeks after induction ALA100 mg/kg was injected intravenously, and diagnostic laparotomy was performed 1, 3, 6, or 9 hours thereafter. MEASUREMENTS AND MAIN RESULTS: The peritoneal cavity was illuminated with the Wood's lamp (ultraviolet light). Fluorescence was determined by direct visualization and compared with a calibrated fluorescent disk. Tissues were collected, sectioned, and examined by fluorescence and conventional light microscopy. Within 1 to 3 hours after intravenous injection of ALA, in vivo fluorescence of tumor nodules (diameter 0.4-5.0 mm) was macroscopically visible. Tumor-free peritoneum did not show fluorescence and was significantly distinguishable from cancer nodules. Fluorescence from intestinal tissues was comparable with tumor nodules. Microscopic fluorescence analysis showed similar values for tumor nodules and peritoneum. Stained histologic specimens of peritoneal surface revealed a superficial layer of cancer cells responsible for fluorescence. The time course of the fluorescence curve in the intestine peaked twice, at 1 and 6 hours after ALA injection. Macroscopically fluorescing nodules were histology confirmed as malignant. CONCLUSIONS: Fluorescence detection of small cancer nodules after intravenous injection of ALA is feasible for nodules smaller than 0.5 mm on the peritoneum. One to 3 hours after drug injection is optimal for diagnosis of metastases.

Aminolevulinic Acid↗

PubMed1997

Non-invasive measurements of breast tissue optical properties using frequency-domain photon migration.

A multiwavelength, high bandwidth (1 GHz) frequency-domain photon migration (FDPM) instrument has been developed for quantitative, non-invasive measurements of tissue optical and physiological properties. The instrument produces 300 kHz to 1 GHz photon density waves (PDWs) in optically turbid media using a network analyser, an avalanche photodiode detector and four amplitude-modulated diode lasers (674 nm, 811 nm, 849 nm, and 956 nm). The frequency of PDW phase and amplitude is measured and compared to analytically derived model functions in order to calculate absorption, mu a, and reduced scattering, mu s, parameters. The wavelength-dependence of absorption is used to determine tissue haemoglobin concentration (total, oxy- and deoxy- forms), oxygen saturation and water concentration. We present preliminary results of non-invasive FDPM measurements obtained from normal and tumour-containing human breast tissue. Our data clearly demonstrate that physiological changes caused by the presence of small (about 1 cm diameter) palpable lesions can be detected using a handheld FDPM probe.

Adult↗

PubMed1997

In vivo fluorescence detection of ovarian cancer in the NuTu-19 epithelial ovarian cancer animal model using 5-aminolevulinic acid (ALA).

The purpose of this study was to determine whether in vivo fluorescence detection of protoporphyrin IX (PpIX) could be used to identify intraperitoneal micrometastases of epithelial ovarian carcinoma after application of 5-aminolevulinic acid (ALA). ALA was applied intraperitoneal at different concentrations (25, 50, and 100 mg/kg) and iv (100 mg/kg) to immunocompetent Fischer 344 rats bearing a syngeneic epithelial ovarian carcinoma. At different time intervals after ALA administration (1.5, 3, and 6 hr) the peritoneal cavity was illuminated with ultraviolet (uv) light. In vivo fluorescence of PpIX initially was determined by direct visualization. Subsequently ex vivo measurements were made with a slow-scan, thermoelectrically cooled CCD camera. Red in vivo fluorescence was observed in ovarian micrometastases smaller than 0.5 mm in 100% of the ALA-administered animals independent of time interval, drug concentration, or route of administration. The intensity of the fluorescence was concentration dependent as strong fluorescence was consistently found only above 25 mg/kg ALA. Ex vivo tumor to peritoneum fluorescence yield peaked 3 hr after administration of a 100 mg/kg intraperitoneal dose. Direct visualization of in vivo fluorescence after ALA application may improve the detection of intraperitoneal ovarian cancer micrometastases.

Aminolevulinic Acid↗

PubMed1997

Time-resolved and steady-state fluorescence measurements of beta-nicotinamide adenine dinucleotide-alcohol dehydrogenase complex during UVA exposure.

beta-nicotinamide adenine dinucleotide (NADH)-alcohol dehydrogenase complex was compared to either UVA irradiation (364 nm; 50 mW cm-2; 0-60 min) or heat in order to investigate complex stability. Prior to irradiation, frequency-domain fluorescence lifetime measurements indicated the presence of two principal components having short (subnanosecond) and long (nanosecond) fluorescence lifetimes reflecting free and bound NADH respectively. UVA exposure resulted in decreased NADH fluorescence intensity concomitant with decreased absorption at 337 nm. However, UVA irradiation did not reduce the fractional contribution of the long-lived bound NADH. The photoinduced fluorescence decrease appeared to be caused by the formation of oxidized NAD+ and not on UVA-induced dissociation of the NADH-protein complex. Such dissociation, detected by a red-shifted fluorescence maximum and decreased fractional contribution of the nanosecond component, was observed when NADH-protein mixtures were heated.

Alcohol Oxidoreductases↗

PubMed1996

Fluorescence imaging studies for the disposition of daunorubicin liposomes (DaunoXome) within tumor tissue.

Unilamellar liposomes that retain their contents in the systemic circulation can alter the pharmacokinetics of anticancer agents in favorable ways. It has long been recognized that certain liposome compositions may increase the local drug concentration substantially above that achievable with a free drug. We report here that liposomes can alter the in vivo disposition of an entrapped drug not only on a macroscopic but also on a microscopic scale. We show through in vitro studies that intact liposomes composed of distearoylphosphatidylcholine and cholesterol and containing daunorubicin (DaunoXome) are taken up into P1798 tumor cells. These liposomes produce an enhanced cytotoxicity relative to the free drug for incubation times longer than about 8 h. For in vivo studies, we developed and used a noninvasive fluorescence imaging technique to follow the accumulation of liposomal daunorubicin within murine tumors. With this method, we show that the maximum concentration of the available liposomal drug in tumors exceeds that of the free drug, and additionally, liposomal daunorubicin persists at high levels for several days. Total liposome-delivered drug fluorescence from whole tumor extracts peaks at about 8 h. In comparison, the fluorescence intensity of daunorubicin demonstrate persistent high levels of daunorubicin fluorescence within cells and throughout the tumor masses. Free daunorubicin, in contrast, transiently achieves modest levels of fluorescence and rapidly drops to background within a few h. These results indicate distinct mechanisms for the localization of free and liposomal daunorubicin, suggesting that liposmal daunorubicin can provide sustained intracellular levels of the drug within the tumor.

Animals↗

PubMed1996

Dosimetry model for photodynamic therapy with topically administered photosensitizers.

BACKGROUND AND OBJECTIVE: Photodynamic therapy (PDT) based on topical application of photosensitizers has been under development over the last years. Typical applications are treatment of basal cell carcinoma of the skin and photoablation of the endometrium. The dosimetry for topically administered photosensitizers must take a time-dependent inhomogenous drug distribution into account together with the conventional parameters such as optical scattering, absorption, and photobleaching. STUDY DESIGN/MATERIALS AND METHODS: This study presents a dosimetry model where the cytotoxic dose is calculated in a stepwise procedure. The first step calculates the time-dependent distribution of 5-aminolevulinic acid (5-ALA) from diffusion theory. In skin this distribution is dependent on drug permeability through the stratum corneum, on the diffusivity of dermis and epidermis, on the drug clearance time, and on the conversion rate from 5-ALA to protoporphyrin IX (PpIX). In the second step the distribution of PpIX is calculated from the 5-ALA distribution found in the first step taking the dynamics of the biosynthesis of 5-ALA to PpIX and the clearance time of PpIX into account. In the third step the generation of cytotoxic singlet oxygen is calculated from the optical distribution during irradiation, taking a photobleaching mechanism into account. RESULTS: The distribution of cytotoxic oxygen is predicted from the optical dose, the drug dose, and the time between the application of the drug and the irradiation. CONCLUSION: The presented dosimetry model is made as simple as possible, yet composite enough to enable all relevant parameters to be taken into account. The model that is based on a linear theory in a semi-infinite medium can, if required, be extended to take nonuniform and nonlinear phenomena into account.

Administration, Topical↗

PubMed1996

Photosensitization of the rat endometrium following 5-aminolevulinic acid induced photodynamic therapy.

BACKGROUND AND OBJECTIVE: The impact of photodynamic therapy (PDT) on the endometrium following topical application of 5-aminolevulinic acid (ALA) was studied in a rat model. Study Design/ MATERIALS AND METHODS: Fluorescence microscopy revealed peak ALA to protoporphyrin IX (Pp IX) conversion 3-6 hours after application. Significantly higher Pp IX levels were observed in the endometrial glands compared with endometrial stroma and myometrium. RESULTS: Histological studies showed PDT-induced endometrial destruction with atrophy 7-10 weeks after treatment. Reproductive performance studies demonstrated significant implantation failure in the treated uterine horns compared with controls. The number of implantation sacs in the treated and untreated horns was 0.4 +/- 0.3 and 8.9 +/- 1.0, respectively (P < 0.01). CONCLUSION: We conclude that the high rate of Pp IX conversion in the endometrial glands can be exploited to treat dysfunctional uterine bleeding with PDT. In addition, this concept may also be applied to study endometrial regeneration and embryo implantation mechanisms.

Administration, Topical↗

PubMed1996

Selective photosensitizer localization in the human endometrium after intrauterine application of 5-aminolevulinic acid.

OBJECTIVE: Our purpose was twofold: to determine the distribution of the endogenous photosensitizer protoporphyrin IX in the uterus and to ascertain the time interval leading to maximal endometrial fluorescence after intrauterine instillation of 5-aminolevulinic acid. STUDY DESIGN: One milliliter of a 400 mg/ml 5-aminolevulinic acid-Hyskon solution was instilled into the uterine cavity of 27 women before hysterectomy. On frozen sections of uterine samples 5-aminolevulinic acid-induced fluorescence was measured with fluorescence microscopy. RESULTS: 5-Aminolevulinic acid-induced fluorescence could first be detected in the superficial endometrial glands 75 minutes after drug injection. In the endometrial gland stumps fluorescence intensity peaked 4 to 8 hours after 5-aminolevulinic acid instillation and was > 48 times higher than in the underlying myometrium. CONCLUSIONS: Fluorescence in the endometrial glands suggests that selective photodynamic destruction of the endometrium may be possible 4 to 8 hours after intrauterine 5-aminolevulinic acid instillation.

Aminolevulinic Acid↗

PubMed1996

Structural and functional effects of endometrial photodynamic therapy in a rat model.

OBJECTIVE: Our purpose was to determine the optical dose required for irreversible endometrial destruction and prevention of implantation by photodynamic therapy with topical 5-aminolevulinic acid. STUDY DESIGN: Three hours after drug application 74 female Sprague-Dawley rats received varying doses of 630 nm of light delivered by an intrauterine cylindric diffusing fiber. RESULTS: A 64 J/cm2 in situ optical dose resulted in long-term irreversible endometrial destruction; 43 J/cm2 damaged endometrial stroma and myometrium but not glandular epithelium 1 day after photodynamic therapy. At this lower light dose endometrium regenerated to full thickness within 3 weeks; however, implantation sacs were significantly reduced. CONCLUSIONS: Photodynamic destruction of glandular epithelium accompanies irreversible endometrial ablation, whereas isolated stromal damage leads to reproductive impairment only. The optical dose required for endometrial ablation is approximately 1.5-fold higher than for reproductive impairment (functional damage) because of differential cell photosensitivity.

Aminolevulinic Acid↗

PubMed1996

Selective photosensitizer distribution in vulvar condyloma acuminatum after topical application of 5-aminolevulinic acid.

OBJECTIVE: Our purpose was to determine the feasibility of selective photosensitization of vulvar condylomas by use of tropical application of 5-aminolevulinic acid. STUDY DESIGN: In vivo fluorescence was assessed and biopsy specimens of condylomas were taken for fluorescence microscopy in 24 patients at different times after application of 2.5% 5-aminolevulinic acid ointment or 20% 5-aminolevulinic acid cream. RESULTS: Both in vivo fluorescence imaging and fluorescence microscopy showed selective fluorescence of condylomas of the labia minora and vestibule only within short time intervals, because fluorescence of poorly keratinized normal epithelium was induced by both 5-aminolevulinic acid formulations. In non-hair-bearing skin, lesional fluorescence remained highly selective. Fluorescence microscopy showed that 90 minutes after drug application peak selectivity in epithelial lesional fluorescence was significantly higher with 2.5% 5-aminolevulinic acid ointment (4.5 +/- 0.9) than it was with 20% cream (2.1 +/- 0.2). CONCLUSION: Selective fluorescence of vulvar condyloma acuminatum can be induced by nonselective topical 5-aminolevulinic acid application. Studies evaluating selective photodynamic destruction of condylomas are justified.

Administration, Topical↗

PubMed1996

Physiological monitoring of optically trapped cells: assessing the effects of confinement by 1064-nm laser tweezers using microfluorometry.

We report the results of microfluorometric measurements of physiological changes in optically trapped immotile Chinese hamster ovary cells (CHOs) and motile human sperm cells under continuous-wave (CW) and pulsed-mode trapping conditions at 1064 nm. The fluorescence spectra derived from the exogenous fluorescent probes laurdan, acridine orange, propidium iodide, and Snarf are used to assess the effects of optical confinement with respect to temperature, DNA structure, cell viability, and intracellular pH, respectively. In the latter three cases, fluorescence is excited via a two-photon process, using a CW laser trap as the fluorescence excitation source. An average temperature increase of < 0.1 +/- 0.30 degrees C/100 mW is measured for cells when held stationary with CW optical tweezers at powers of up to 400 mW. The same trapping conditions do not appear to alter DNA structure or cellular pH. In contrast, a pulsed 1064-nm laser trap (100-ns pulses at 40 microJ/pulse and average power of 40 mW) produced significant fluorescence spectral alterations in acridine orange, perhaps because of thermally induced DNA structural changes or laser-induced multiphoton processes. The techniques and results presented herein demonstrate the ability to perform in situ monitoring of cellular physiology during CW and pulsed laser trapping, and should prove useful in studying mechanisms by which optical tweezers and microbeams perturb metabolic function and cellular viability.

2-Naphthylamine↗

PubMed1996

A mathematical model for light dosimetry in photodynamic destruction of human endometrium.

We are involved in the development of photodynamic therapy (PDT) as a minimally invasive method for treating dysfunctional uterine bleeding, one of the primary clinical indications for hysterectomy. In this paper, we analyse light propagation through the uterus in order to specify the requirements for a light delivery system capable of effectively performing endometrial PDT. Our approach involves developing an analytical model based on diffusion theory to predict optical fluence rate distributions when cylindrical and spherical optical applicators are placed in the uterine cavity. We apply the results of our model calculations to estimate the thermal effects of optical irradiation and the effective photodynamic optical dose. Theoretical fluence rate calculations are compared to fluence rate measurements made in fresh, surgically removed human uteri. Our results show that a trifurcated cylindrical optical applicator inserted into the human uterus can provide a light dose that is sufficient to cause photodynamic destruction of the entire endometrium. When the optical power per unit length of each cylindrical applicator is 100 mW cm-1 (at 630 nm), a fluence rate of 40 mW cm-2 is delivered to the boundary layer between the endometrium and the myometrium (a depth of about 4-6 mm). The optical fluence delivered to the boundary layer after 20 min of exposure is 50 J cm-2, a level that is generally accepted to cause tissue damage throughout the endometrium in most patients.

Dose-Response Relationship, Radiation↗

PubMed1996

Effects of ultraviolet exposure and near infrared laser tweezers on human spermatozoa.

Photostress has to be considered during optical micromanipulation of gametes. Ultraviolet light, including low-energy UVA (32-400 nm) radiation, as well as high-intensity near infrared (NIR) laser radiation may induce cell damage. A total number of 580 light-exposed sperm cells were studied in single-cell photostress experiments. Low-power (1.5 mW, 5.3 W/cm2) UVA exposure with 365 nm radiation of a standard mercury microscopy lamp to human spermatozoa resulted within 109 +/- 30 s in paralysis and within 310 +/- 110 s in cell death. Cytotoxic effects during cell manipulation with laser microbeams were found to be partly based on non-linear excitation phenomena, in particular two-photon absorption by endogenous cell chromophores. Two-photon absorption will be more intense in the case of pulsed laser microradiation, but occur also during micromanipulation with highly focused continuous wave (cw) microbeams used as laser tweezers ('optical traps'). In particular, short-wavelength NIR traps < 800 nm induce UVA-like biological effects (oxidative stress). For example, sperm trapping with 760 nm microbeams resulted in UVA-like autofluorescence modifications, paralysis within 35 +/- 20 s and cell death within 65 +/- 20 s. In contrast, laser microbeams at 800-1064 nm may act as relatively safe micromanipulation tools. In most optical traps multifrequency cw lasers are employed. Radiation of these lasers can magnify cytotoxic effects. Therefore, single-frequency laser operation should be preferred. In general, laser assisted cell micromanipulation requires a new understanding of microbeam-cell interaction, including aspects of non-linear optics.

Cell Survival↗

PubMed1996

Regeneration processes in rabbit endometrium: a photodynamic therapy model.

The origin and process of regeneration in rabbit endometrium was evaluated following photodynamic epithelial destruction using topically applied aminolevulinic acid (ALA). Selective destruction of endometrial epithelium was performed using photodynamic therapy (PDT). ALA was diluted to 200 mg/ml dextran 70 shortly prior to administration. A volume of 1.2 ml was injected into the left uterus. Intrauterine illumination (wavelength 630 nm, light dose 40-80 J/cm2) was performed 3 h after drug administration. Tissue morphology was evaluated by light and scanning electron microscopy 1, 3, 7 and 28 days post-treatment (three animals at each time-point). Regeneration of the endometrium following epithelial ablation by PDT was fully activated after 24 h and was completed after 72 h. Endometrial surface generation occurred by proliferation, originating primarily in deeper regions of the glands. Findings from our morphological follow-up study support the origin of endometrial regeneration being mainly from undifferentiated stem cells and residual glandular epithelium.

Aminolevulinic Acid↗

PubMed1996

Determination of motility forces of human spermatozoa using an 800 nm optical trap.

The measurement and calculation of trapping forces on ellipsoidal specimens, and the determination of intrinsic motility forces of human spermatozoa by employing an 800 nm optical trap ("laser tweezers"), are presented. ATP-driven motility forces were calculated from calibrated trapping forces generated during the interaction of an 800 nm laser beam with single sperm cells. Calibration was performed by moving optically trapped sperm heads through a laminar fluid and calculating viscous forces for an ellipsoidal cell shape. Sperm heads were obtained by microsurgically removing flagellum with a pulsed laser beam ("laser scissors"). A trapping efficiency of 0.12 +/- 0.02 and a mean intrinsic motility force of 44 +/- 20 pN were determined for motile spermatozoa from healthy donors.

Humans↗