Tissue Fluorophores Research Articles

Polarization Sensing of Fluorophores in Tissues for Drug Compliance Monitoring

We used a new method, polarization sensing, to monitor the concentration of the fluorophore rhodamine 800 in an intralipid suspension and in chicken tissue. Rhodamine 800 (Rh800) could be excited at 648 nm using a laser pointer. We developed a simple device for measuring the combined emission from a highly polarized reference film and the unpolarized or orthogonally polarized emission of Rh800 from the scattering intralipid or tissue. The concentration of Rh800 in this medium was revealed by large changes in the polarization (P) with values of P ranging from 0.8 to −0.9. It is possible to vary the sensitive Rh800 concentration range by variation of the detected emission wavelengths, orientation of the excitation polarizer, or fluorophore concentration in the reference film. Polarization sensing of fluorophores in tissue requires only steady-state detection, and can be accomplished with simple and/or portable electronics. Such devices may find use in electronic detection of ingested medicines based on transdermal detection of nontoxic long-wavelength fluorophores.

Modulation Sensing of Fluorophores in Tissue: A New Approach to Drug Compliance Monitoring

We describe a method to detect the presence of fluorophores in scattering media, including intralipid suspensions and chicken muscle covered with skin. The fluorophores were rhodamine 800 (Rh800) and indocyanine green (IcG), both of which can be excited at long wavelengths where there is minimal absorption by tissues. These fluorophores were dissolved in intralipid or in chicken muscle under skin. A method to approximate the fluorophore concentration in such samples was developed using a long lifetime reference fluorophore in a polymer film placed immediately on the illuminated surface of the sample. Because of the long lifetime of the reference film, the modulation of its emission at low frequencies near 2 MHz is near zero. Since the lifetime of Rh800 and IcG are below 2 ns the modulation of the combined emission is a measure of the intensity of the fluorophore (Rh800 or IcG) relative to the long lifetime reference. Using this method we were able to measure the concentration-dependent intensities of Rh800 and IcG in an intralipid suspension. Additionally, micromolar concentrations of these probes could be detected in chicken muscles, even when the muscle was covered with a layer of chicken skin. The presence of an India ink absorber in the intralipid had only a moderate effect on the modulation values. We suggest the use of this transdermal detection of long-wavelength fluorophores as a noninvasive method to monitor patient compliance when taking medicines used for treatment of chronic diseases such as AIDS or tuberculosis. © 1999 Society of Photo-Optical Instrumentation Engineers.

Emission spectra of colonic tissue and endogenous fluorophores.

Autofluorescence emission spectra of normal, adenomatous, and malignant tissues of the colon were compared to that of known fluorophores to indicate the possible causes of tissue fluorescence. Data were collected from normal mucosa (n = 18), adenomatous polyps (n = 32), and adenocarcinoma (n = 18) of the colon. A range of cellular and extracellular fluorophores (elastin, collagen, flavin adenine dinucleotide, nicotinamide adenine dinucleotide, phenylalanine, pyridoxal 5' phosphate, tryptophan, and tyrosine) were similarly examined using a spectrofluorometer with emission and excitation spectrometers. Emission intensities were plotted against wavelength. Wavelengths of peak emission and the width of each peak at half its maximum intensity were measured. Colonic tissue gave four major emission peaks, the wavelengths of which were independent of tissue histology. Tryptophan and collagen type IV appeared to be responsible for two of the peaks. It is possible that NADH may be the cause of a third emission maxima.

Emission Spectra of Colonic Tissue and Endogenous Fluorophores

Autofluorescence emission spectra of normal, adenomatous, and malignant tissues of the colon were compared to that of known fluorophores to indicate the possible causes of tissue fluorescence. Data were collected from normal mucosa (n = 18), adenomatous polyps (n = 32), and adenocarcinoma (n = 18) of the colon. A range of cellular and extracellular fluorophores (elastin, collagen, flavin adenine dinucleotide, nicotinamide adenine dinucleotide, phenylalanine, pyridoxal 5' phosphate, tryptophan, and tyrosine) were similarly examined using a spectrofluorometer with emission and excitation spectrometers. Emission intensities were plotted against wavelength. Wavelengths of peak emission and the width of each peak at half its maximum intensity were measured. Colonic tissue gave four major emission peaks, the wavelengths of which were independent of tissue histology. Tryptophan and collagen type IV appeared to be responsible for two of the peaks. It is possible that NADH may be the cause of a third emission maxima.

Influence of tumour depth, blood absorption and autofluorescence on measurements of exogenous fluorophores in tissue

We investigated the influence of tumour depth and differences in blood concentration and autofluorescence between tumour and normal tissue on the fluorescence of a tumour-localising agent. Carotenoporphyrin, CP(Me)3, was injected into rats and nude mice with intradermal tumours. On the tumours an incision was made, uncovering 2 mm2 of the tumour, and fluorescence measurements, including excitation-emission maps and fluorescence ratios, were made on skin, covered and uncovered tumour. The measured fluorescence ratio in the uncovered tumour showed a three- to tenfold increase compared to the covered tumour. We used a one-dimensional layered tissue model to analyse the data. In conclusion, even with a high tumour-selectivity deeper lying tumours cannot always be detected, particularly if the tumour has a high blood concentration or low autofluorescence intensity.

Propagation of fluorescent light

Background and Objective In general, the remitted fluorescence spectrum is affected by the scattering and absorption properties of tissue. Other important factors are boundary conditions, geometry of the tissue sample, and the quantum yield of tissue fluorophores. Each of these factors is examined through a series of Monte Carlo simulations. Study Design/Materials and Methods Monte Carlo modeling is used to simulate the propagation of excitation light and the resulting fluorescence. Remitted fluorescence is determined for semi-infinite single and multiple layer geometries and for cubic geometries representing small tissue samples. Monte Carlo results are compared to approximations obtained with a heuristic model. Results Remitted fluorescence as a function of (1) the depth of fluorescence generation and (2) radial escape position is presented for semi-infinite single and multiple layer geometries. Fluorescence from a small tissue sample is simulated in terms of a cubic geometry, and losses from the sides and bottom are presented as a function of cube dimensions in terms of optical depth of the excitation wavelength. Monte Carlo results for a homogeneous semi-infinite layer are compared to a simple, fast heuristic model. Conclusion Both Monte Carlo simulations and the heuristic model clearly detail the volume of tissue interrogated by fluorescence. Since ∼ 35–40% of the remitted fluorescence is due to photons originally directed away from the surface, distal layers affect the remitted fluorescence. Fluorescence spectra from small biopsy samples may not produce the correct line shape owing to wavelength dependent losses. Lasers Surg. Med. 21:166–178, 1997. © 1997 Wiley-Liss, Inc.

Propagation of fluorescent light.

In general, the remitted fluorescence spectrum is affected by the scattering and absorption properties of tissue. Other important factors are boundary conditions, geometry of the tissue sample, and the quantum yield of tissue fluorophores. Each of these factors is examined through a series of Monte Carlo simulations. Monte Carlo modeling is used to simulate the propagation of excitation light and the resulting fluorescence. Remitted fluorescence is determined for semi-infinite single and multiple layer geometries and for cubic geometries representing small tissue samples. Monte Carlo results are compared to approximations obtained with a heuristic model. Remitted fluorescence as a function of (1) the depth of fluorescence generation and (2) radial escape position is presented for semi-infinite single and multiple layer geometries. Fluorescence from a small tissue sample is simulated in terms of a cubic geometry, and losses from the sides and bottom are presented as a function of cube dimensions in terms of optical depth of the excitation wavelength. Monte Carlo results for a homogeneous semi-infinite layer are compared to a simple, fast heuristic model. Both Monte Carlo simulations and the heuristic model clearly detail the volume of tissue interrogated by fluorescence. Since approximately 35-40% of the remitted fluorescence is due to photons originally directed away from the surface, distal layers affect the remitted fluorescence. Fluorescence spectra from small biopsy samples may not produce the correct line shape owing to wavelength dependent losses.

Fluorophore quantitation in tissue-simulating media with confocal detection

Fluorescence measurements from tissue are increasingly being used as a medical diagnostic procedure to assess tissue malignancy or tissue function. Unfortunately, the reemitted fluorescent intensity measured from a tissue surface is not necessarily proportional to the fluorophore concentration because the light is altered by the tissue's intrinsic absorption and scattering properties. By measuring fluorescence from tissue volumes which are smaller than the average scattering length, the effects of the tissue's intrinsic absorption are diminished. In this study, experiments with tissue simulating phantoms are used, as well as Monte Carlo simulations of the experiment, to demonstrate the utility of point fluorescence detection for diagnostic measurements. Potential applications of this technique range from photosensitizer quantitation in vivo, pharmacokinetic measurements of fluorophore in different tissues, to any application where fluorophore quantification is required from a highly scattering medium.

Fluorescence lifetime-based sensing in tissues: a computational study

We have numerically solved the photon diffusion equation to predict the distribution of light in a tissue model system with a uniform concentration of fluorophore. Our results show that time-dependent measurements of light propagation can be used to monitor the fluorescent lifetimes of a uniformly distributed fluorophore in tissues. With proper referencing, frequency-domain measurements of phase-shift, theta, may allow quantitation of fluorescent lifetimes, tau, independent of changes in the local absorption and scattering properties. These results point to a new approach for noninvasive diagnostic monitoring through quantitation of fluorescent lifetime, tau, when the lifetime of the fluorophore is comparable with photon migration times.

Future developments in ocular fluorophotometry instrumentation

The scope of ocular fluorometry is to monitor exogenous and endogenous fluorophores in ocular tissues, in relation with ophthalmic and systemic diseases using the unique optical prospectives of the eye. The elderly population and the incidence of blindness are increasing rapidly due to more cases of diabetes, glaucoma, cataract and age-related macular degeneration. Monitoring changes in specific fluorophores in the eye may help identify the high risk groups in these diseases. New developments in instrumentation include differential fluorometry and introduction of confocal optics. Differential fluorometry has already achieved significant progress for the study of the autofluorescence of the lens and cornea and measurements in the aqueous. Improved spatial resolution obtained with improved optics opens interesting possibilities like measurement of corneal endothelial permeability and retinal vascular permeability. The results already obtained will be presented with particular incidence on measurements of lens fluorescence (normals--336.2 +/- 56.3; diabetes--659.9 +/- 123.9; age group--40-50 y) and corneal endothelial permeability (normals--3.14 +/- 60.10(-1) cm-1).

Alteration of spectral characteristics of human artery wall caused by 476-nm laser irradiation.

Fluorescence spectroscopy is a promising new technique for discrimination of normal and atherosclerotic arterial tissues. It has been suggested that this technique be used as a guidance system for laser angiosurgery catheters; however, irradiation by 476-nm light can change the spectroscopic properties of arterial tissue. We present studies that establish intensity levels and exposure times at which alterations in tissue spectral properties are minimal. We also investigate the nature of spectral alterations following exposure of normal human aorta to high intensities of 476-nm laser light. Changes in laser-induced fluorescence (LIF) are characterized by two prominent features: the peak fluorescence intensity decreases permanently, and the fluorescence lineshape changes in a largely reversible way. We relate these changes to alterations in individual tissue chromophores: permanent changes in absolute fluorescence intensity are due to irreversible changes in tissue fluorophores, reversible changes in fluorescence lineshape are due alterations in tissue absorbers. A simple kinetic model is used to describe the decrease in absolute fluorescence intensity.

Fluorescent, physiological and pharmacokinetic properties of fluorescein glucuronide

When fluorescein is administered systemically, it is metabolized to form a fluorescent compound, fluorescein monoglucuronide. The molar fluorescent intensity of this compound is less than that of fluorescein but its fluorescent intensity compared to fluorescein varies considerably depending on the wavelength employed for excitation. Several hours after oral or intravenous administration the conjugate can become the dominant fluorophore in the plasma. When fluorescein is administered systemically and fluorescent intensity is measured in the eye, it is necessary to determine which compound is being measured since the time courses of the plasma concentrations and the permeability of the ocular barrier of each compound may not be the same. A technique suitable for measuring the relative concentration of each fluorophore in ocular tissues in vivo is suggested.