WO2001003571A1 - Method of measuring concentration of luminescent materials in turbid media - Google Patents
Method of measuring concentration of luminescent materials in turbid media Download PDFInfo
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- WO2001003571A1 WO2001003571A1 PCT/CA2000/000823 CA0000823W WO0103571A1 WO 2001003571 A1 WO2001003571 A1 WO 2001003571A1 CA 0000823 W CA0000823 W CA 0000823W WO 0103571 A1 WO0103571 A1 WO 0103571A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue
- A61B5/1455—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue using optical sensors, e.g. spectral photometrical oximeters
- A61B5/14551—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue using optical sensors, e.g. spectral photometrical oximeters for measuring blood gases
- A61B5/14556—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue using optical sensors, e.g. spectral photometrical oximeters for measuring blood gases by fluorescence
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
- A61B5/0071—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence by measuring fluorescence emission
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
- A61B5/0082—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence adapted for particular medical purposes
- A61B5/0084—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence adapted for particular medical purposes for introduction into the body, e.g. by catheters
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B2562/00—Details of sensors; Constructional details of sensor housings or probes; Accessories for sensors
- A61B2562/02—Details of sensors specially adapted for in-vivo measurements
- A61B2562/0233—Special features of optical sensors or probes classified in A61B5/00
- A61B2562/0242—Special features of optical sensors or probes classified in A61B5/00 for varying or adjusting the optical path length in the tissue
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue
- A61B5/1455—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue using optical sensors, e.g. spectral photometrical oximeters
- A61B5/1459—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue using optical sensors, e.g. spectral photometrical oximeters invasive, e.g. introduced into the body by a catheter
Definitions
- the present invention relates to a method and device for measuring concentrations of luminescent materials in turbid media, and more particularly the invention relates to measurement of fluorophore concentrations in turbid media such as tissue in vivo using a combined fluorescence/reflectance measurement technique.
- tissue Light directed into a turbid medium undergoes two phenomena; scattering and absorption.
- the amount of scattering is determined by the tissue structure such as cell and mitochondria size, while the absorption is determined by the quantity of endogenous absorbers such as melanin and porphyrins (e.g. hemoglobin in blood).
- endogenous absorbers such as melanin and porphyrins (e.g. hemoglobin in blood).
- melanin and porphyrins e.g. hemoglobin in blood.
- Different tissue types scatter and absorb light in different amounts, i.e. liver versus muscle.
- Many recent pharmacokinetic studies are using fluorescent drugs to monitor body processes.
- new forms of cancer treatments use fluorescent drugs. These treatment methods require accurate knowledge of the drug concentration for proper treatment.
- a possible method of determining the concentration of the fluorescent drug is to measure its fluorescence.
- the strength of the fluorescence signal will depend on the intensity of excitation light, and the scattering and absorption properties of the turbid medium.
- U.S. Patent No. 4,178,917 to Shapiro discloses a method and system for non-invasive detection of zinc protoporphyrins using an excitation beam and two detectors, one being termed the reference detector for measuring scattered or reflected light S 2 at the excitation wavelength.
- the fluorescence detector measures the fluorescence S 1 from the red blood cells flowing through the measurement volume.
- the intent of this device therefore, is to measure only the concentration of fluorophores within the blood stream and not in the surrounding tissue.
- the signal is influenced by the intervening tissues and the method described does not correct for differences in these tissues.
- a further drawback to this method is that it is not readily adaptable for measuring fluorophore concentrations in a wide range of turbid media.
- a method of measuring concentration of a luminescent compound in a turbid medium comprising: illuminating a turbid medium with a beam of light having an effective wavelength ⁇ ., to excite luminescence in a luminescent compound being detected in the turbid medium; measuring a luminescence signal F an effective distance of about D1 from the beam of light; measuring a reflectance signal R at wavelength ⁇ ., an effective distance of about D2 from the beam of light; and processing the measured R and F signals to produce an effective function f(F, R) and comparing f(F, R) to a calibration curve of f c (F c , R c ) versus concentration of the luminescent compound in a turbid medium to determine a concentration of the luminescent compound.
- a method of determining pairs of distances D1 and D2 for measuring luminescence and reflectance respectively in order to reduce effects of scattering and absorption variations between turbid samples, the distances D1 and D2 being measured from a beam of light used to induce luminescence in one or more luminescent compounds in the turbid medium comprising the steps of; a) providing an effective reference turbid media having optical properties mimicking the turbid media and adding known amounts of a luminescent compound so as to increase the concentration of the luminescent compound and after addition of each known amount exciting the reference turbid medium with a beam of light at an effective wavelength and measuring a luminescence signal at a plurality of distances D1 from the beam of light and measuring a reflectance signal at a plurality of distances D2 from the beam of light; b) repeat step a) for an effective number of reference turbid media possessing a range of optical properties; c) for each pair of distances, plot an effective function of both lumi
- the present invention also provides a device for measuring concentration a light source for producing a beam of light of wavelength ⁇ .,; first detector means for measuring fluorescence; second detector means for measuring light of wavelength ⁇ and a holder for holding the light source, first and second detector means, the holder including a planar portion adapted to be placed on a surface of a tissue and an adjustment mechanism for adjusting a distance between the light source and the first detector and a distance between the light source and the second detector.
- Figure 1a is a diagrammatic representation showing the relative relationship between excitation source and detectors for measuring reflectance and fluorescence according to the method of the present invention
- Figure 1 b shows an interstitial embodiment in which the fibers are located in the turbid medium
- FIG 2 is a block diagram showing an apparatus for measuring fluorophore concentrations in turbid media constructed in accordance with the present invention
- Figure 3a shows the raw fluorescence signal for various concentrations of
- AIPcS 4 measured in a series of turbid solutions of different optical properties with the fluorescence being excited at 670 nm and measured at 720 nm a distance of 0.86 mm from the source fiber;
- Figure 3b is a plot of the ratio of the fluorescence of AIPcS 4 measured at 0.86 mm from Figure 3a to the reflectance of the excitation light measured at
- Figure 4a is a plot of the raw fluorescence signal for fluorescein excited at 488 nm, with fluorescence measured at 530 nm a distance of 0.86 mm from the source fiber;
- Figure 4b is a plot of the ratio of fluorescence of fluorescein measured at 0.86 mm from Figure 4a to the reflectance of the excitation light measured at
- Figure 5a is a plot of the raw fluorescence of AIPcS 4 measured at 750 nm excited at 633 nm versus concentration of AIPcS 4 ;
- Figure 5b is a plot of the ratio of fluorescence of AIPcS 4 collected at 0.86 mm (depicted in Figure 5a) to reflectance collected at 1.42 mm versus AIPcS 4 concentration, the reflectance measured at the excitation wavelength, 633 nm;
- Figure 6a is a plot of the raw fluorescence of AIPcS 4 in a series of turbid solutions measured at 680 nm excited at 633 nm versus concentration of AIPcS 4 ;
- Figure 6b is a plot of the ratio of fluorescence of AIPcS 4 collected at 0.86 mm (depicted in Figure 6a) to reflectance collected at 1.42 mm versus AIPcS 4 concentration, the reflectance measured at the excitation wavelength, 633 nm;
- Figure 7a is a plot of the normalized reflectance collected at 0.86 mm and 1.42 mm from the source fiber versus the background absorption coefficient, the reflectance measured at the excitation wavelength, 633 nm;
- Figure 7b is a plot of the normalized AIPcS 4 fluorescence collected at
- Figure 8 is a plot of the measured fluorescence/reflectance ratio of AIPcS 4 versus background absorption of the turbid medium, the fluorescence was measured at 680 nm and 750 nm and collected at 0.86 mm and the reflectance was measured at 633 nm and collected at 1.42 mm;
- Figure 9 is a plot of the measured fluorescence/reflectance ratio for AIPcS4 versus background absorption of the turbid medium for other possible pairs of fluorescence and reflectance collection distances, the fluorescence was measured at 750 nm and the reflectance was measured at 633 nm;
- Figure 10 is a plot of the measured fluorescence/reflectance ratio AIPcS 4 versus reduced scattering coefficient of the turbid medium for several combinations of fluorescence distance and reflectance collection distances with the fluorescence measured at 750 nm and reflectance measured at 633 nm, the concentration of AIPcS 4 was 1 ⁇ g/ml and background absorption of the turbid
- Figure 11 is a contour plot of percentage standard deviation of F/R normalized to the drug absorption coefficient for a fixed fluorophore concentration versus D1 and D2 as determined by Monte Carlo calculations;
- Figure 13 is a plot of concentrations determined by the non-invasive fluorescence technique versus the concentration determined by fluorescence assay in three different tissue types for a rabbit injected with AIPcS 4 ;
- Figure 14a is a plot of the raw fluorescence at 750 nm collected at both
- Figure 14b is a plot of the ratio of fluorescence AIPcS 4 collected at 0.86 mm from Figure 10a to the reflectance of the excitation light collected at 1.42 mm versus the AIPcS 4 concentration as determined by tissue assay with the fluorescence measured at 750 nm and the reflectance measured at the excitation wavelength, 633 nm.
- the preferred embodiment of the method of the present invention uses a fluorescence plus reflectance technique to measure quantitatively the in vivo concentration of a fluorescent drug or photosensitizer (PS), (both referred to hereinafter as fluorophores) independent of the tissue optical properties.
- PS fluorescent drug or photosensitizer
- the basic principle involves using the reflectance measurement as a means of correcting for tissue scattering and absorption in the fluorescence measurement, in practice, a non-invasive probe is placed on the tissue surface, and light directed to the tissue by means of an optical fiber. This light is used to excite the fluorophores. Fluorescence from the fluorophores is measured via another fiber at a known distance (D1 ) from the excitation fiber, while the excitation light is simultaneously collected at a another distance (D2).
- D1 known distance
- D2 another distance
- the excitation light measured at D2 is referred to as the reflectance R since it is due to light at the excitation wavelength undergoing scattering/reflection within the body of the tissue and is not referring to specular reflectance from the surface of the tissue. Therefore, as defined herein the term "reflectance" is being used even though the quantity may be measured in the interior of the turbid medium rather than at its surface.
- the inventors have made the unexpected finding that, to a first approximation, the correct choice of D1 reduces the effect of scattering and the correct choice of D2 corrects for the reduction in fluorescent intensity that would accompany an increase in tissue absorption.
- the distances D1 and D2 are relative to the excitation position on the surface of the turbid medium and are independent of each other. Therefore, D1 and D2 define the radii of two circles about the excitation or source position upon which the collection fibers may be positioned.
- the excitation light is directed into the tissue at an angle substantially perpendicular to the surface of the tissue, while the fluorescence and reflectance light are collected substantially perpendicular to the surface.
- the light may also be directed into the tissue at an angle other than normal incidence and collected at angles other than those normal to the surface.
- the distances D1 and D2 may be different than those for the case where the illumination and collection light is normal to the surface.
- Figure 1 b shows another embodiment of the invention.
- three fibers are used; one to deliver excitation light to the medium, one to collect the fluorophore fluorescence at a distance D1 and one to collect the excitation light at a distance D2.
- This embodiment differs from the one described above in that now the three fibers are placed in the tissue so that while the device used in Figure 1a may be considered non-invasive, the device in Figure 1b may be invasive.
- the distances D1 and D2 are likely to be different from that used in the non-invasive embodiment described above, but there will be a choice of D1 and D2 such that the ratio of fluorescence/reflectance is a constant for fixed concentration of fluorophore.
- Figure 1 b shows the ends of the three fibers placed at the same depth in the turbid medium. In practice, there would be no restriction on the relative vertical placement of the three fibers.
- the ratio of fluorescence/reflectance will be approximately constant, regardless of the optical properties of the tissue.
- a calibration curve is made by measuring fluorescence and reflectance for a range of fluorophore concentrations in a variety of turbid liquid samples and plotting the ratio F/R as a function of concentration of the fluorophore (luminescent material). A user can then make a measurement of a tissue sample, compare the ratio to that of the calibration curve, and estimate the in vivo concentration.
- Device 10 for measuring concentration of fluorophores in a turbid medium 12.
- Device 10 includes a light source 14 for delivering excitation light at wavelength A., to the surface of the turbid medium 12 (i.e. tissue).
- the excitation light at wavelength K ⁇ is delivered from source 14 to turbid medium 12 by an optical fiber 16 which is coupled to source 14 at one end and at the other end is held in probe 18 at an angle perpendicular to the face of the probe.
- the probe is positioned in contact with the surface of a patient's skin.
- Another optical fiber 22 is placed a distance of 1.30 to 1.70 mm away from fiber 16 and is held in the probe at angle perpendicular to the face of the probe.
- Fiber 22 is connected to a detector 24.
- An adjustment mechanism 17 provides for adjustment of the distance D1 between the distal end portion of fiber 22 located adjacent to the tissue surface with respect to the distal end portion of source fiber 16 located adjacent to the tissue surface.
- the purpose of fiber 22 is to collect the excitation light at A., that has traveled through the turbid medium 12 and back to the surface of the medium. This signal is called the reflectance, R. Since both reflectance and fluorescence signals will be collected by fiber 22, separation of the reflectance signal at A., from the fluorescence signals is necessary and may be achieved in any one of several ways.
- band pass filter 26 that allows only light at A., to pass through to the detector 24
- monochromator (not shown) set to allow only light at A., to pass through to the detector
- combination of band pass filter and monochromator not shown
- spectrometer and detector array such as a photodiode array or charge-coupled device array
- spectrometer and detector array such as a photodiode array or charge-coupled device array
- Another optical fiber 30 is placed a distance of 0.55 to 0.95 mm away from fiber 16 and is held in the probe at an angle perpendicular to the face of the probe.
- the end face of fiber 30 is non-reflecting.
- An adjustment mechanism 19 provides for adjustment of the distance D2 between the distal end portion of fiber
- Fiber 30 located adjacent to the tissue surface with respect to the distal end portion of source fiber 16.
- Fiber 30 is connected to a detector 32 and the purpose of fiber 30 is to collect the fluorescence at ⁇ 2 that has traveled through the turbid medium and back to the surface of the medium. This signal is called the fluorescence, F, since typically this light is due to fluorescence of the fluorophores excited at A.,. Since both reflectance and fluorescence will be collected by fiber 30, separation of the fluorescence from the reflectance may be achieved in one of several ways.
- band pass filter 34 that allows only light at ⁇ 2 to pass through to the detector 32
- monochromator (not shown) set to allow only light at ⁇ 2 to pass through to the detector
- combination of band pass filter and monochromator not shown
- spectrometer and detector array such as a photodiode array or charge-coupled device array
- a combination of band pass filter, spectrometer and detector array such as a photodiode array or charge-coupled device array.
- the ends of the excitation fiber 16 and collection fibers 22 and 30 are positioned in the probe so that they are perpendicular to the fiber holder 18 and hence perpendicular to the tissue surface. If the fibers are positioned at different angles, then the distances noted in the description above for D1 and D2 will be different. These distances can be determined by following the procedure described hereinafter.
- the signal output from detector 24 and the signal output from detector 32 are connected to a signal processor 40 for calculating the ratio F/R.
- the optical fibers 16, 22 and 30 used are typically 100 to 400 ⁇ m in diameter.
- the excitation light at A. is preferably selected to match the electronic absorption band of the fluorophore.
- the light source 14 may be a low intensity laser at A.,, a light emitting diode at A.,, or a white light lamp source, with a band pass filter (not shown) placed along the delivery path such that only light at ⁇ 1 is delivered to the surface of turbid medium 12.
- the detectors 24 and 32 may include but are not restricted to be a photodiode detector, a charge coupled device (CCD) detector or a photomultiplier tube (PMT).
- the excitation light at ⁇ 1 excites a fluorophore in the turbid medium such that it produces fluorescence at a second wavelength, ⁇ 2 .
- the fluorophores are typically exogenous to the turbid medium.
- the fluorophore may be a fluorescent drug in tissue.
- the ratio of the collected signals, F/R is then used to determine the concentration of the exogenous fluorophore in the turbid medium by comparing the measured ratio to that of a calibration curve of concentration versus F/R.
- the calibration curve of f c (F c , R c ) versus concentration of the fluorophore is determined by measuring the fluorophore fluorescence in a turbid liquid sample, in which the concentration of the fluorophore can be controlled and varied by the addition of small aliquots of the fluorophore to the turbid sample. Measurements of the ratio F/R on the sample at different fluorophore concentrations then gives a simple plot of F/R versus concentration.
- calibration measurements only need to be made on one turbid sample. In practice, they may be made on a series of turbid liquid samples that have a variety of scattering and absorption properties. Doing so increases the certainty that the correct calibration curve has been generated.
- a significant advantage of the present method and device over the prior art is achieved by the presence of fiber 22 and measuring the ratio F/R which minimizes the changes in fluorescence signal due to differences in scattering and absorption properties between different samples.
- the distances noted above have been found to be the best match for removing the scattering and absorption variations. Other distances have been tried but have not been found to be as effective.
- the method of the present invention advantageously reduces the effect of signal variations due to variations in excitation light source intensity without elaborate signal processing or excitation light monitoring techniques and it reduces the effect of variations in the fluorescence signal due to measuring on different tissue types.
- the present invention provides a measurement of the in vivo concentration of a fluorophore in a turbid medium regardless of the scattering and absorption properties of the medium.
- An advantage of the method is that the measurement can be made non-invasively from the surface of the turbid medium so that in for example medical applications, this method can be used for non-invasively measuring in vivo drug concentrations without the use of biopsies.
- the present method may be readily used for the measurement of the concentration of a luminescent material, or more particularly, a fluorophore in blood.
- the three fibers may be incorporated into a catheter with the probes spaced apart the appropriate distance at the end of the catheter which is insertable into a blood vessel.
- the method of measuring concentration of luminescent materials in turbid media can also be used in non biomedical applications.
- Several nonlimiting examples include measurement of luminescent or fluorescent materials in bioreactors that typically comprise turbid suspensions of cells and the like; measurement of pigments in paints and concentration of pigments and other labeled ingredients in plastics.
- the present method may also be used for studying flow processes in turbid media by attaching luminescent/fluorescent labels to species and studying the flow and mixing behavior in the turbid media.
- the optimum distances D1 and D2 are obtained by constructing several sample turbid media with known optical properties mimicking the optical properties of the samples for which the concentration measurements are to be made. Fluorescence and reflectance measurements are then performed at several distances from the excitation point for all turbid samples and a best fit to f(F, R) is obtained for each pair of distances. The pairs of distances exhibiting the lowest sum of squares of residuals are preferred for making the measurements since the lower sum of squares of residual values indicates that a measurement of the ratio of the fluorescence to the reflectance at those distances is substantially indicative of the fluorophore concentration in the turbid sample, regardless of the optical properties of the sample.
- Example 2 discussed hereinafter illustrates the experimental approach to determining the preferred values of D1 and D2.
- D1 and D2 may also be calculated theoretically using a model such as Monte Carlo simulation, numerical solution of the radiation transport equation, or diffusion theory to calculate the fluorescence at a plurality of distances D1 from the source and the reflectance at a plurality of distances D2 from the source for a fixed concentration of fluorophore and for certain values of the absorption and scattering coefficients of the turbid medium.
- the calculation is repeated for a range of increasing fluorophore concentrations. This is repeated for an effective number of turbid media with an appropriate range of optical properties. For each pair of distances the effective function of both fluorescence and reflectance (f(F,
- the water-based phantom solutions typically consisted of intralipid as the scatterer, Melan ink as background absorber and one of AIPcS 4 , or Fluorescein as the fluorescent drug being measured.
- Intralipid phantoms have been used as tissue-simulating materials for several years by many researchers, see for example "Light scattering in Intralipid 10% in the wavelength range of 400 -1100 nm", H.J. van Staveren et al., Applied Optics 30: 4507 - 4514 (1991 ).
- the apparatus consisted of a diffuse reflectance and fluorescence probe, spectrometer, and CCD imaging system.
- the baseline optical properties of the liquid intralipid phantoms were determined using the general diffuse reflectance technique of Patterson et al. disclosed in T.J. Farrell, M.S. Patterson, and B.C. Wilson "A diffusion theory model of spatially resolved steady state diffuse reflectance for the noninvasive determination of tissue optical properties in vivo", Med. Phys. Vol 19, pp 879-888 (1992); M.S.
- the probe consisted of a source, and 8 collection fibers located 0.86 to 10 mm from the source fiber along the circumference of a 10 mm circle. At the spectrometer entrance, the collection fibers were equally spaced parallel to the entrance slit axis. The spectrometer disperses the light from each fiber onto the CCD detector. One exposure of the CCD therefore provides spectra of light collected at eight distances over a range of 300 nm with a resolution of 2 nm. Fluorescence measurements were made by replacing the broad band QTH lamp as the light source with a laser at an appropriate wavelength for the excitation of the drug being studied.
- the plot in Figure 3b depicts the ratio of the fluorescence measured at 0.86 mm to the reflectance of the excitation light measured at 1.42 mm versus the drug concentration. Note that data points from different phantom solutions fit on almost the same line. As can be observed in the graph of Figure 3b, the fluorescence/reflectance ratio is essentially constant for a constant fluorophore concentration in "tissues" of different optical properties.
- Simple arithmetic can then be used to quantify the fluorophore concentration of the sample being measured. If the plot of F/R is nonlinear, then calibration needs to be made over the whole range of concentration values to be measured. In this case, a simple look-up table is sufficient for quantifying the fluorophore concentration of the measured sample.
- a further series of intralipid phantom measurements were made with the intent of determining the optimal pair of distances to be used in the fluorescence/reflectance ratio.
- a series of liquid phantoms was prepared using intralipid as the scatterer and Melan ink as the absorber.
- the optical properties of the phantoms were determined using spatially resolved diffuse reflectance.
- Several phantoms of different optical properties were prepared.
- the fluorophore AIPcS 4 was added in known aliquots so as to increase the concentration of the fluorophore in the phantom.
- a measurement was taken of the reflectance and fluorescence at several distances (0.86 mm to 3.4 mm respectively).
- the ratio of fluorescence to reflectance was calculated for all twenty-five combinations of distances. For example, the ratio of fluorescence to reflectance was calculated for fluorescence at 0.86 mm to reflectance at 0.86 mm, 1.42 mm, 1.92 mm, 2.38 mm and 3.4 mm. This procedure was repeated for fluorescence measured at 1.42 mm, 1.92 mm, 2.38 mm and 3.4 mm from the source fiber. This measurement and calculation was performed for four sets of phantoms, each with different background optical properties.
- Figure 5a shows the raw fluorescence at 750 nm collected at 0.86 mm for the 4 different phantoms measured. Note that the fluorescence at each concentration varies substantially depending on the background optical properties.
- Figure 5b is the same set of measurements, but it depicts the ratio of the fluorescence at 0.86 mm to the reflectance at 1.42 mm. A linear regression of this data gives an R 2 value of 0.9822.
- Tables 1 and 2 give the R 2 values for all sets of distance pairs for the fluorescence measured at 680 nm and 750 nm. For both fluorescence wavelengths, the optimal pair of distances is fluorescence at 0.86 mm and reflectance at 1.42 mm. The set of background optical properties used in these experiments is typical of the optical properties measured for tissue.
- Figure 7b shows the normalized fluorescence measured at 0.86 mm and 1.42 mm from the source fiber over absorption coefficients of 0.001 - 1.0 mm '1 .
- the reduced scattering coefficient was equal to 1.0 mm '1 and the AIPcS 4 concentration was 1 ⁇ g/ml.
- the measured signal drops substantially with increased background absorption. It is also apparent that the changes in reflectance and fluorescence signals are different at the two measurement distances.
- Figure 8 shows the ratio of the fluorescence measured at 0.86 mm to the reflectance measured at 1.42 mm for both 750 nm and 680 nm versus background absorption. Clearly, the ratio remains the same across the large range of background absorption values, indicating that for a fixed concentration of fluorophore, the ratio of fluorescence to reflectance with this pair of distances is constant, despite large changes in background absorption.
- Figure 9 the ratio of fluorescence to reflectance versus absorption is shown for the other possible distance combinations. For these other distance pair combinations, the ratio changes significantly with increasing absorption, even though the concentration of fluorophore remains constant.
- EXAMPLE 4 Effect of Reduced Scattering on the Ratio of Fluorescence to Reflectance
- Figure 10 shows the fluorescence/reflectance ratios for several combinations of fiber distances.
- the combinations that give the smallest change with increased reduced scatter are 0.86/1.42 and 1.42/1.42, both with standard deviations of only 5% or less.
- the pair at 0.86/1.42 has a slightly smaller standard deviation than the collection pair at 1.42/1.42, although it is uncertain if the differences in the standard deviations for these two sets of data is statistically significant.
- Example 3 demonstrates that the selection of the correct collection distances is important in reducing the effects of scattering on the F/R measurement, although this selection may not be unique.
- the measured F/R has a smaller standard deviation across the range of physiologically important tissue scattering values than most other combinations measured.
- a rabbit was injected with AIPcS 4 at a dose of 3 mg/kg and measurements taken 24 hours later. Measurements of the fluorescence and reflectance were taken on the back skin, the underlying muscle and, ex vivo, on the liver. These tissue types were chosen because of their large range of optical properties. The measured tissue was excised and the drug uptake determined by fluorescence assay as described in L. Lilge, C. O'Carroll and B.C. Wilson "Photosensitizer quantification for ex vivo tissue samples: a tissue solubilization technique", J. Photochem. Photobiol. B, Vol 39 pp 229- 235 (1997).
- a rabbit was injected with AIPcS 4 at a dose of 1.5 mg/kg and measurements taken 24 hours later. Measurements were taken on several locations on the skin, and muscle of the rabbit, as well as two positions on the liver.
- FIG. 14a shows the raw fluorescence measured at both 0.86 mm and 1.42 mm and the ratio of fluorescence measured at 0.86 mm to the reflected excitation light at 1.42 mm is shown in Figure 14b.
- the raw fluorescence measured on the skin is more than twice that measured on the liver, even though the in vivo concentration in the liver is twice that of the skin.
- the much lower fluorescence signal measured in the liver tissue is due to its much higher absorption coefficient than in skin.
- Clearly, relying on the fluorescence signal alone gives an inaccurate assessment of the in vivo drug concentration, since the different optical properties of different tissue types affects the amount of fluorescent light reaching the tissue surface.
- Table 1 Correlations of measured ratio to concentration for several fluorescence/reflectance distance pairs (mm). Fluorescence measure at 680nm, reflectance measured at 633 nm.
- Table 2 Correlations of measured ratio to concentration for several fluorescence/reflectance distance pairs (mm). Fluorescence measured at 750 nm, reflectance measured at 633 nm.
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EP00945506A EP1206211A1 (en) | 1999-07-13 | 2000-07-13 | Method of measuring concentration of luminescent materials in turbid media |
CA002375760A CA2375760A1 (en) | 1999-07-13 | 2000-07-13 | Method of measuring concentration of luminescent materials in turbid media |
AU59598/00A AU5959800A (en) | 1999-07-13 | 2000-07-13 | Method of measuring concentration of luminescent materials in turbid media |
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US (1) | US6219566B1 (en) |
EP (1) | EP1206211A1 (en) |
AU (1) | AU5959800A (en) |
CA (1) | CA2375760A1 (en) |
WO (1) | WO2001003571A1 (en) |
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US8082016B2 (en) | 2001-05-22 | 2011-12-20 | Alfred E. Mann Institute For Biomedical Engineering At The University Of Southern California | Measurement of cardiac output and blood volume by non-invasive detection of indicator dilution |
EP1981401A2 (en) * | 2006-01-20 | 2008-10-22 | Alfred E. Mann Institute for Biomedical Engineering at the University of Southern California | Measurement of cardiac output and blood volume by non-invasive detection of indicator dilution |
EP1981401A4 (en) * | 2006-01-20 | 2010-07-21 | Alfred E Mann Inst Biomed Eng | Measurement of cardiac output and blood volume by non-invasive detection of indicator dilution |
EP2070469A1 (en) * | 2007-12-10 | 2009-06-17 | FUJIFILM Corporation | Image processing system, image processing method, and program |
US8260016B2 (en) | 2007-12-10 | 2012-09-04 | Fujifilm Corporation | Image processing system, image processing method, and computer readable medium |
Also Published As
Publication number | Publication date |
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CA2375760A1 (en) | 2001-01-18 |
AU5959800A (en) | 2001-01-30 |
EP1206211A1 (en) | 2002-05-22 |
US6219566B1 (en) | 2001-04-17 |
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