WO2000022414A1 - Caracterisation de la luminescence dans un milieu de dispersion - Google Patents

Caracterisation de la luminescence dans un milieu de dispersion Download PDF

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Publication number
WO2000022414A1
WO2000022414A1 PCT/US1999/023709 US9923709W WO0022414A1 WO 2000022414 A1 WO2000022414 A1 WO 2000022414A1 US 9923709 W US9923709 W US 9923709W WO 0022414 A1 WO0022414 A1 WO 0022414A1
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WIPO (PCT)
Prior art keywords
light
wavelength
medium
luminophore
emission
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PCT/US1999/023709
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English (en)
Inventor
Eva M. Sevick-Muraca
Ralf H. Mayer
Jeffery S. Reynolds
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The Texas A & M University System
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Application filed by The Texas A & M University System filed Critical The Texas A & M University System
Priority to AU11099/00A priority Critical patent/AU1109900A/en
Priority to EP99954855A priority patent/EP1127262A4/fr
Priority to CA2346728A priority patent/CA2346728C/fr
Priority to US10/473,303 priority patent/US7054002B1/en
Publication of WO2000022414A1 publication Critical patent/WO2000022414A1/fr
Priority to NO20011791A priority patent/NO20011791L/no

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/47—Scattering, i.e. diffuse reflection
    • G01N21/4795—Scattering, i.e. diffuse reflection spatially resolved investigating of object in scattering medium

Definitions

  • the present invention relates to spectroscopic techniques involving luminescence, and more particularly, but not exclusively relates to the determination of lifetime of a luminophore in a light scattering medium.
  • Fluorescent probes in the near-infrared range appear particularly promising for in vivo biomedical diagnostic techniques that involve external, noninvasive measurements or minimally invasive, endoscopic measurements of emitted light.
  • quantitative lifetime measurements for such probes are often difficult to obtain in the light scattering environment typically encountered with in vivo diagnostics.
  • Light scattering also hampers other applications of luminophore probes both inside and outside the biomedical field. Consequently, lifetime measurements are usually restricted to dilute, nonscattering solutions. Notably, even equipment used in this manner, such as a curvette to contain the dilute solution, tends to scatter light to some degree introducing an attendant inaccuracy.
  • one form of the present invention is a unique technique to evaluate a medium including a luminophore.
  • Other forms include unique systems and methods to measure optical properties of a luminophore in a light scattering medium.
  • a light scattering medium includes a luminiphore that is exposed to a number of different wavelengths. Multiply scattered light from exposure to these wavelengths is measured, and one or more optical characteristics of the medium are determined relative to the different wavelengths.
  • an optical characteristic of a luminophore in a light scattering medium is determined.
  • the medium is illuminated by light at a first wavelength corresponding to an emission wavelength of the luminophore and at a second wavelength corresponding to an excitation wavelength of the luminophore. Multiply scattered light in response to illumination by the different wavelengths is detected to optically characterize the medium and/or luminophore.
  • a further form of the present invention is a technique to determine the lifetime of a fluorophore. This technique includes both a method and instrumentation directed to fluorescence lifetime measurements. Preferably, this technique does not utilize a reference fluorophore and is not adversely impacted by any light scattering and absorption that might occur in a scattering medium.
  • Still a further form of the present invention includes a frequency-domain approach to measuring fluorescence lifetime of one or more fluorophores.
  • This approach may include exciting a fluorophore in a light scattering sample with a modulated excitation light and detecting phase shift information.
  • the fluorescence lifetime may be determined from the phase shift information through relationships characterizing light scattering by the sample.
  • a light scattering sample containing a fluorophore is exposed to an intensity modulated light at a first wavelength selected to cause the fluorophore to fluoresce at a second wavelength of light. Scattered light at the first wavelength is detected and scattered light at the second wavelength is detected. Optical properties are determined from the detected scattered light to provide fluorescence lifetime based on relationships that characterize photon migration in a light scattering medium. This form may include characterizing the detected scattered light in terms of phase shift in the frequency domain.
  • a system of the present invention includes an intensity modulated excitation light source configured to deliver an excitation light of a first wavelength to a light scattering substance containing a fluorophore of interest.
  • the fluorophore responds to the excitation light to provide a light emission at a second wavelength.
  • Scattered light is detected at two locations spaced apart from one another.
  • a processor gathers information corresponding to the detected scattered light and processes this information to determine fluorescence lifetime by applying relationships that characterize photon migration of scattered light.
  • a first detector may be used for detection of light at a first one of the locations that includes a first optical fiber coupled to a first sensor.
  • a second detector may be used for detection of light at a second one of the locations that includes a second optical fiber coupled to a second sensor.
  • the first and second detectors may also include corresponding first and second optical filters to selectively detect the first wavelength of light with the first sensor and the second wavelength of light with the second sensor.
  • the coupling arrangement of the first and second fibers to the first and second sensors may be interchanged to obtain comparative measurements for the minimization of equipment inaccuracies.
  • FIG. 1 is a schematic view of a system according to one embodiment of the present invention.
  • Figs. 2A and 2B depict a flow chart illustrating one process that may be performed with the system shown in Fig. 1.
  • Fig. 3 is a schematic view of a system of another embodiment of the present invention.
  • Fig. 4 depicts normalized excitation and emission spectra for DTTCI (top) and ICG (bottom) in a comparative format pertinent to experimental examples.
  • Fig. 5 plots phase shift versus modulation frequency for two different concentrations of DTTCI in a comparative format pertinent to experimental examples.
  • Fig. 6 plots phase shift versus modulation frequency for two different concentrations of ICG in a comparative format pertinent to experimental examples.
  • Fig. 7 plots lifetime versus modulation frequency for DTTCI and ICG corresponding to the plots of Figs. 5 and 6.
  • Fig. 8 plots relative modulation attenuation versus modulation frequency for two different concentrations of DTTCI in a comparative format pertinent to experimental examples.
  • Fig. 9 plots relative modulation attenuation versus modulation frequency for two different concentrations of ICG in a comparative format pertinent to experimental examples.
  • Fig. 10 plots lifetime versus modulation frequency for DTTCI and ICG corresponding to the plots of Figs. 8 and 9.
  • lifetime refers to the mean survival time of an activated luminophore or the mean time between the absorption of an excitation photon and emission of a photon.
  • multiply scattered light refers to light that travels at least five (5) times the mean isotropic scattering length [(l-g) ⁇ s ] " '; where g is the mean cosine of angular scatter and ⁇ s is the scattering coefficient of the medium.
  • Fig. 1 illustrates evaluation system 20 of one embodiment of the present invention.
  • System 20 includes light source instrumentation 30, container 40, detection instrumentation 50, and processor 70.
  • Light source instrumentation 30 includes modulation signal generator 22 having a range of selectable output Radio Frequencies (RF).
  • Generator 22 drives two monochromatic light sources in the form of laser diodes 24, 26.
  • Laser diodes 24, 26 are intensity modulated at a selected RF frequency ( ⁇ ) input from generator 22 to provide light at an excitation wavelength ( ⁇ x ) and emission wavelength ( ⁇ m ), respectively.
  • Instrumentation 30 also includes kinematic mirror 32 that is operable to successively select between the two laser beams at wavelengths ⁇ x , ⁇ m output by the respective laser diodes 24, 26.
  • Light from mirror 32 encounters continuously variable neutral density filter wheel 34 of instrumentation 30 to selectively adjust intensity.
  • Lens assembly 36 of instrumentation 30 collects the light output by neutral density filter wheel 34 for input to optical source fiber 38.
  • Source fiber 38 enters container 40.
  • Container 40 holds a light scattering medium M including a selected amount of a luminophore as a constituent.
  • Interchangeable connectors 46a, 46b couple fibers 44a, 44b to detector 48 of detection instrumentation 50.
  • Detector 48 includes two optic channels 50a, 50b coupled by connectors 46a, 46b to receive light from detector fibers 44a, 44b, respectively.
  • Each channel 50a, 50b has a corresponding interchangeable/removable interference filter (IF) 54a, 54b; adjustable neutral density filter (NDF) 56a, 56b; and light sensor 58a, 58b.
  • IF interchangeable/removable interference filter
  • NDF adjustable neutral density filter
  • Light sensors 58a, 58b are coupled to processor 70 to provide one or more output signals corresponding to light detected from medium M.
  • Sensors 58a, 58b are arranged in a standard heterodyne configuration and may be of any form such as Photomultiplier Tubes (PMTs), photodiodes, or image intensified Charge Coupled Devices (CCDs) to name just a few.
  • PMTs Photomultiplier Tubes
  • CCDs image intensified Charge Coupled Devices
  • RF signal generator 60 is phase locked to generator 22 at a slightly different frequency ⁇ + ⁇ as represented by coupling 62; where ⁇ is the frequency difference.
  • generator 60 The output of generator 60 is amplified by RF amplifier 64 and mixed at sensors 58a, 58b to provide a corresponding differential output signal from which information corresponding to phase and modulation magnitude of the detected light can be determined with processor 70. Accordingly, processor 70 is also operatively coupled to generators 22, 60.
  • Processor 70 includes a port for insertion and removal of a portable memory device such as an electromagnetically or optically encoded disk, cartridge, or tape.
  • processor 70 is also operatively coupled to visual display 76 and one or more Input/Output (I/O) devices 78, including for example a keyboard, mouse, light pen, acoustic loudspeakers, microphone, and/or printer just to name a few.
  • I/O Input/Output
  • Processor 70 may be comprised of one or more components configured as a single unit, or when of a multi- component form, processor 70 may have one or more components remotely located relative to the others, or otherwise have its components distributed throughout system 20.
  • Processor 70 may be programmable, a state logic machine or other type of dedicated hardware, or a hybrid combination of programmable and dedicated hardware.
  • One or more components of processor 70 may be of the electronic variety including digital circuitry, analog circuitry, or both.
  • processor 70 may include one or more optical elements.
  • Processor 70 includes an integrated and/or remote storage capability in the form of one or more types of memory. By way of nonlimiting example, this memory may include one or more of the solid-state, magnetic, and/or optical memory types.
  • Such memory types may include Random Access Memory (RAM), Sequential Accessible Memory (SAM) (such as the First-In, First-Out (FIFO) variety, or the Last-In, First-In LIFO variety), Programmable Read Only Memory (PROM), Electrically Programmable Read Only Memory (EPROM), flash memory or Electrically Erasable Programmable Read Only Memory (EEPROM); an optical disc memory (such as a CD ROM); a magnetically encoded hard disc, floppy disc, tape, or cartridge; another variety of storage device as would occur to those skilled in the art, or a combination of any of these types.
  • RAM Random Access Memory
  • SAM Sequential Accessible Memory
  • PROM First-In, First-Out
  • EPROM Electrically Programmable Read Only Memory
  • EEPROM Electrically Erasable Programmable Read Only Memory
  • an optical disc memory such as a CD ROM
  • magnetically encoded hard disc, floppy disc, tape, or cartridge another variety of storage device as would occur to those skilled in the art, or a combination of any of these types.
  • the memory may be volatile, nonvolatile, or a hybrid combination of volatile and nonvolatile varieties.
  • memory may be permanently installed, in a portable form that may be readily removed and reinstalled, or a combination of these types.
  • processor 70 is of a standard personal computer configuration with a common solid-state digital integrated processing unit operatively coupled to solid-state memory.
  • appropriate interfaces are installed to facilitate control of generators 22, 60 and receipt of data from detector 48.
  • the memory of this embodiment contains programming to be executed by the processing unit, and is arranged for reading and writing of data in accordance with one or more routines executed by processor 70.
  • processor 70 may include any oscillators, control clocks, interfaces, signal compensators/conditioners, filters, limiters, Analog-to-Digital (A/D) converters, Digital- to- Analog (D/A) converters, communication ports, or other types of circuits as would occur to those skilled in the art to implement the present invention.
  • Processor 70 is configured to execute one or more routines to perform selected calculations with data received from detector 48 for lifetime evaluation process 120.
  • evaluation process 120 utilizes Frequency Domain Photon Migration (FDPM) techniques to extract the characteristic optical properties of medium M. These properties are obtained for photons of two wavelengths: the wavelength used to optically excite a selected luminophore and the wavelength of the luminescence resulting from this excitation. Lifetime measurements are determined from these wavelength-dependent characterizations of the medium and comparative luminescence measurements, as described hereinafter.
  • FDPM Frequency Domain Photon Migration
  • a luminiphore probe with known excitation wavelength ⁇ t and emission wavelength ⁇ m is selected for evaluation and placed in light scattering medium M.
  • This light scattering medium M may include, for example, living biologic tissue for which metabolites/analytes are being interrogated in terms of lifetime of the selected luminophore probe.
  • the light scattering medium M may be a cell culture, flow cytometry stream, a chemical reaction medium or other light scattering environment as would occur to those skilled in the art.
  • endogenous luminophores in medium M are interrogated without introduction of an exogenous probe in stage 122.
  • stage 130 intensity modulated light at frequency ⁇ with the excitation wavelength ⁇ x of the designated luminophore is provided by light source instrumentation 30 to source site 39 of medium M. Accordingly, light from laser diode 24 is provided from light source instrumentation 30 through fiber 38 to site 39. The illuminating light is subsequently scattered and/or absorbed by medium M. As multiply scattered light sourced from site 39 reaches sites 42a, 42b; it can be sensed with detection instrumentation 50 and quantitized in terms of frequency domain parameters of relative phase shift and/or modulation magnitude. Filters of detector 48 are removed and/or adjusted to facilitate detection of the excitation light wavelength with sensors 58a, 58b for this stage.
  • Photon transport in a light scattering medium M may be modeled as a diffusive process.
  • the photon density U(r, ⁇ ) in a homogeneous medium at a vector position r can be related to optical properties of the medium by the diffusion equation (1) as follows:
  • the magnitude of R( ⁇ , ⁇ ) represents gain and conversion efficiency of the photon transport and the angle represents the phase delay of the modulated light.
  • the absorption coefficient ⁇ a and the isoptropic scattering coefficient ⁇ ' s characterize pertinent light scattering properties of the medium at a given light wavelength.
  • the subscripts ⁇ t x" and "m" are used to designate various optical parameters specific to the excitation and emission wavelengths, respectively.
  • medium M is exposed to modulated light of wavelength ⁇ r at site 39 as sourced from laser diode 24 of light source instrumentation 30.
  • P x ( ⁇ ) is a complex number that represents the source magnitude and phase
  • ⁇ (r) is the Dirac delta function.
  • U x (r, ⁇ ) is the frequency domain excitation photon density in the medium M and the complex wave vector k x ( ⁇ ) is given by expression (3) as follows: x ⁇ c ⁇ ax J
  • the photon density is related to the observed modulation phase ⁇ (r, ⁇ ) by expression (5a) as follows:
  • m x (r, ⁇ ) M x (r, ⁇ ) m 3S m dx (r, ⁇ ) (8)
  • This ratio is generally independent of source modulation.
  • data is collected and stored in processor 70 corresponding to detected multiply scattered light output from medium M. It should be appreciated that only one of relative phase and modulation attenuation information needs to be observed to provide the desired characterization of the medium M at the excitation wavelength.
  • a regression or other iterative estimation can be employed. To facilitate this calculation, operation 132 detects multiply scattered light outputs for a number of different RF modulation frequencies.
  • the effective response function of the detection instrumentation is designated ⁇ mstr .
  • the instrument effects can be removed by taking one half of the difference between these two relative measurements.
  • Measurements gathered with detection instrumentation 50 in operations 132, 134 of stage 130 for a desired number of different modulation frequencies are recorded with processor 70.
  • Stage 140 is next encountered to obtain an optical characterization of medium M at the emission wavelength ⁇ m in terms of abso ⁇ tion and isotropic scattering coefficients ⁇ am , ⁇ ' sm -
  • emission wavelength light is provided to source site 39 with laser diode 26 of light source instrumentation 30, instead of excitation wavelength light from laser diode 24.
  • Filtering of detector 48 is removed/adjusted to facilitate detection of multiply scattered light output from medium M at the emission wavelength ⁇ m and measurement of relative modulation phase or magnitude over a selected range of modulation frequencies in the manner described for stage 130.
  • the emission photon density is characterized in stage 140 in accordance with expressions (2)- (9) substituting the emission wavelength for the excitation wavelength (and correspondingly substituting subscript "x" with "m” in these expressions).
  • operation 144 the measurements are repeated with coupling of site 42a being switched to sensor 58b of channel 50b and coupling of site 42b being switched to sensor 58a of channel 50a.
  • the measurements of operations 142, 144 are recorded with processor 70 to determine abso ⁇ tion and isotropic scattering coefficients ⁇ am , ⁇ ' sm -
  • stage 150 luminescence data is gathered.
  • Excitation wavelength light is provided to site 39 of medium M from laser diode 24 of light source instrumentation 30 in operation 152 of stage 150 and frequency domain measurements are gathered.
  • detection instrumentation 50 is configured with filtering of channel 50a adjusted/removed to detect the excitation wavelength with sensor 58a and filtering of channel 50b adjusted/removed to detect the emission wavelength with sensor 58b.
  • fluorescence photon density may be modeled in accordance with expression (10) as follows (where the subscript "f ' denotes fluorescence optical parameters):
  • the quantum efficiency of the fluorophore is denoted by ⁇ and ⁇ af describes abso ⁇ tion of excitation light due to fluorescence. Fluorescence decay is assumed to be of the monoexponential type with lifetime ⁇ ; however, the principles of the present invention can be applied to multiexponential decays using techniques known to those skilled in the art.
  • expression (3) the fluorescence photon density of expression (10) can be written as presented in the following expression (11): TTt Q ⁇ n) P( ⁇ ) ,_ , , ⁇ , (11)
  • ⁇ (r, ⁇ ) ⁇ (r, ⁇ )/ ⁇ (r, ⁇ ) and determinations of ⁇ (r, ⁇ ) and ⁇ (r, ⁇ ) are based on measured phase shifts ⁇ x ( ⁇ r, ⁇ ) and ⁇ f ( ⁇ r, ⁇ ).
  • the fluorescence photon density is related to fluorescence modulation Mf(r, ⁇ ) by expression (5b).
  • processor 70 performs calculations with the data collected during stages 130, 140, 150.
  • processor 70 performs regression analysis of measurements from excitation characterization stage 130 to determine the abso ⁇ tion and isotropic scattering coefficients U aX , ⁇ ' sx at the excitation wavelength.
  • processor 70 performs regression analysis of measurements from emission characterization stage 140 to determine the abso ⁇ tion and isotropic scattering coefficients ⁇ am , ⁇ ' sm at the emission wavelength.
  • processor 70 calculates lifetime based on the measurements obtained from luminescence characterization stage 150 and the coefficients obtained from operations 162, 164.
  • phase shift ⁇ t ⁇ r, ⁇ ) ⁇ f (r 2 , ⁇ ) - ⁇ x (r ⁇ , ⁇ ) is adjusted by the addition of phase change ⁇ xc (r ⁇ , ⁇ ) associated with the excitation propagation from site 39 to site 42a at ri .
  • This phase value ⁇ xc (r ⁇ , ⁇ ) can be calculated from expression (6a) and the optical characteristics determined in operation 162 from measurements of excitation wavelength characterization stage 130.
  • the modulation magnitude detected with detection instrumentation 50 is given by expression (18) as follows:
  • excitation M xc (r, ⁇ ) and emission M mc (r, ⁇ ) can be determined from expression (7).
  • the product of source modulation and detection instrument response function can be determined from expression (8) at the excitation:
  • stage 160 which depends upon a comparison between measured and calculated modulation information and a constant ratio of modulations of the sources at emission and excitation wavelengths. Varying the source modulation ratio, the lifetime measured at multiple modulation frequencies can be regressed to obtain a unique source modulation ratio associated with a vanishing slope, i.e. minimized ⁇ of the lifetime distribution, and to obtain the resulting lifetime ⁇ . After lifetime is determined in stage 160 from relative phase or modulation measurements, it is output in stage 170. concluding process 120. It should be understood that only one possible sequence of the measurement stages 130, 140, 150 is illustrated. Indeed, the various measurements may be performed in many other sequences with respect to selected light wavelengths, modulation frequencies, and coupling configuration of sites 42a. 42b relative to channels 50a, 50b. Also, the calculations of stage 160 may be performed any time by processor 70 in relation to the measurement stages 130, 140, 150 to the extent measurement data has been provided to processor 70.
  • the arrangement of system 20 may differ.
  • light source instrumentation may include a source of excitation and/or emission wavelength light such as a different type of laser, lamp, or other device as would occur to those skilled in the art.
  • detection instrumentation 50 may include an optical multiplexer controlled by processor 70 to provide for the switching of optical couplings relative to detector 48.
  • the measurements may be based on two spaced apart light source sites 39 in medium M with just one detection site 42a or 42b and corresponding sensor 58a or 58b.
  • light source instrumentation may be configured with another set of the components described for instrumentation 30 to provide two light source channels; while detection instrumentation 50 could be modified to include only a single channel 50a or 50b.
  • the lifetime calculations may be readily adapted to this dual source using techniques known to those skilled in the art. Also, instrument function that might lead to calculation discrepancies relative to the two source sites can be addressed by switching the equipment supplying light to each source; thereby providing the first and second measurement configurations described in connection with stages 130, 140, 150.
  • Fig. 3 depicts medical diagnostic system 220 of another embodiment of the present invention.
  • System 220 includes diagnostic instrument 225 to evaluate a patient's medical condition based on lifetime readings of an endogenous, exogenous, or immobilized exogenous fluorophore in a patient's tissue 280.
  • Instrument 225 includes light source apparatus 230, detection apparatus 250, and processor 270 that are operationally configured like light source instrumentation 30, detection instrumentation 50, and processor 70, respectively, but are packaged in a manner convenient for in vivo interrogation of tissue 280.
  • the fluorophore in tissue 280 is typically an exogenous probe introduced into tissue 280 that has a known excitation and emission wavelength to which the optics of instrument 225 are matched.
  • exogenous probes may be immobilized in a subcutaneous implant, by oral ingestion, or any other means that would occur to those skilled in the art.
  • the present application may be applied to monitor endogenous fluorophores for which excitation/emission wavelengths are known.
  • Light source apparatus 230 includes one or more sources to provide light to tissue
  • the source light is provided to site 239 of tissue 280 as represented by arrow IL.
  • the source light is multiply scattered by tissue 280 and received by detection apparatus at sites 242a, 242b as represented by arrows MSI, MS2.
  • Instrument 225 is configured to maintain a generally constant relative spacing between sites 239, 242a, and 242b, corresponding to r 0 , ri, and r 2 , respectively.
  • Processor 270 may be configured to control selection of the appropriate source light wavelength and modulation frequencies for light source apparatus 230 and the corresponding filter configuration for detection apparatus 250 to automatically perform the stages and operations of process 120 with instrument 225.
  • instrument 225 may include a moveable hand-held wand or optical probe coupled to a base unit; where the wand is arranged for placement proximate to surface 282 of tissue 280.
  • surface 282 may be the patient's skin, with instrument 225 being arranged to make percutaneous measurements.
  • instrument 225 may include an endoscope coupled to a base unit that is arranged to perform interrogations through a body lumen, cavity, or small surgical incision.
  • surface 282 can be the boundary of the body lumen or an organ selected for interrogation, to name just a few examples.
  • the calculations performed in stage 160 may be adjusted to account for finite boundary conditions to improve performance.
  • instrument 225 is arranged to place sites 239, 242a, 242b in the tissue, more closely approximating infinite boundary conditions.
  • instrument 225 may be completely contained in a portable device that is battery powered.
  • Yet another embodiment includes interrogating a light scattering medium including an amount of a selected luminophore with light at an emission wavelength of the luminophore and sensing multiply scattered light at the emission light wavelength in response to this interrogation to provide a first optical characterization of the medium.
  • the medium is also exposed to light at an excitation wavelength of the luminophore and a multiply scattered luminescence at the emission wavelength is detected in response to this exposure.
  • a value corresponding to lifetime of the luminophore is determined from the first optical characterization and the luminescence.
  • a further embodiment includes exposing a light scattering medium including an amount of a luminophore to a number of different light wavelengths selected relative to the luminophore.
  • a plurality of optical characteristics of the medium are established by sensing multiply scattered light at each of the different wavelengths.
  • a value corresponding to lifetime of the luminophore is determined from the optical characteristics and a multiply scattered emission at a first one of the light wavelengths caused by exposure of the medium to a second one of the light wavelengths.
  • Still a further embodiment provides for an evaluation of a light scattering medium including an amount of a luminophore with a light interrogation system.
  • This system includes a first device optically coupled to a first site of the medium and a second device optically coupled to a second site of the medium that is spaced apart from the first site. Also included are subjecting the medium to a first light wavelength selected relative to the luminophore, optically coupling the first device to the second site and the second device to the first site, and exposing the medium to the first light wavelength after coupling.
  • a value corresponding to lifetime of the luminophore is determined from a first light output and a second light output.
  • Another embodiment includes means for illuminating a light scattering medium including a luminophore; means for characterizing light scattering behavior of the medium for an excitation wavelength of the luminophore and an emission wavelength of the luminophore; and means for determining lifetime of the luminophore from the characterizing means and a multiply scattered light emission from the medium at the emission wavelength in response to illumination by light at the excitation wavelength. Also, because luminophores may have different activated states, they may have multiple lifetimes corresponding to these states.
  • FDPM measurements according to process 120 were conducted in a light scattering tissue- simulating phantom form of medium M.
  • DTTCI 3,3'-Diethylthiatricarbocyanine Iodide
  • ICG or ER-125 Indocyanine Green
  • Container 40 for the phantom was a cylindrical acrylic vessel 1 1.3 cm in diameter and 15 cm in height.
  • the phantom was prepared as an aqueous (DULF water; Fisher Scientific, Fair Lawn, NJ.) intralipid (20%; Pharmacia & Upjohn Company, Clayton, N.C.) solution to mimic tissue-like scattering properties.
  • a DTTCI stock solution was prepared with ethanol; and ICG was dissolved directly in water. Appropriate quantities of stock solutions were suspended in 1.0% Intralipid to yield the final fluorophore concentrations in the phantoms as listed in Table 1 that follows:
  • Figure 4 shows the concentrations of the analyzed dyes and presents the relevant excitation and emission spectra measured with a spectrofiuorometer (Fluorolog-2; SPEX Industries, Inc., Edison, N.J.). The lifetime of DTTCI dissolved in ethanol was previously reported as 1.33 ⁇ 0.02 ns at 790-nm excitation and 820-nm emission wavelengths.
  • the lifetime of ICG was reported to be 0.57 ⁇ 0.02 ns at 780-nm excitation and 830-nm emission wavelengths measured without scattering by use of time-domain techniques as well as by conventional frequency-domain fluorimetry referenced against the known lifetime of a well- characterized fluorophore.
  • excitation wavelengths were provided by laser diodes of the 56 DFS series; Melles Griot, Boulder, Colorado with power/wavelengths of 3 mW at 749nm for ICG excitation and 25 mW at 778nm for DTTCI excitation.
  • a 30 mW 830 nm laser diode from Melles Griot was used for emission wavelengths.
  • Kinematic mirror 32 was a model 9891 from New Focus, Santa Clara and neutral density filter wheel 34 was from Newport of Irvine. California.
  • Source fiber 38 was a 1000- ⁇ m-core source fiber (3M FT SILICA/0.39-NA TECS multimode fiber type).
  • the light sensed by the fibers was delivered to sensors 58a, 58b in the form of gain-modulated photomultiplier tube (PMT) detectors (Model H6573; Hammamatsu, Bridgewater, N.J.).
  • the detector fibers 44a, 44b were terminated with connectors 46a, 46b in the form of SMA fiber-optic connectors that could be interchanged conveniently to perform the reconfigurations of stages 130, 140, 150.
  • PMT gain-modulated photomultiplier tube
  • Detector 48 included neutral-density filters 56a, 56b from CVI Laser Co ⁇ oration, Albuquerque, N.M. and interference filters 54a, 54b of a narrow-bandpass form (lOnm FWHM; CVI Laser Co ⁇ oration). Also included were lens assemblies that focused the collected light onto the PMTs. The gain settings of the PMTs remained unchanged during all measurements. The three neutral-density filters in the setup aided in maintaining constant dc levels of the detected PMT signals. The filters 34, 54a, 54b were used to adapt the setup to different output power levels of the two source laser diodes 24, 26 and to facilitate acquisition of light intensity signals over a large dynamic range.
  • the PMTs were gain modulated at the modulation frequency of the laser diodes plus an offset frequency of 100 Hz ( ⁇ ).
  • the heterodyned PMT signals were then digitized, Fourier transformed, and analyzed for phase shift and amplitude attenuation by use of Lab VIEW software (National Instruments Co ⁇ oration, Austin, Tex.) executed by a personal computer form of processor 70.
  • the experimental results illustrated in Figs. 5-7 are based on relative phase measurements in stages 130, 140, 150. To obtain these results the stages were performed in a different order than illustrated in the flow chart of Figs. 2A and 2B. Namely, Stage 150 was performed first, followed by stage 130, and then stage 140.
  • the measurement protocol for the lifetime determination comprises the three stages 130, 140, 150 for each concentration; where each stage includes measurements for two different configurations over a range of modulation frequencies from about 60 to about 140 MegaHertz (MHz).
  • the kinematic mirror placed in the experimental setup expedited execution of the three stages of each measurement.
  • Optical parameters of the sample at excitation and emission wavelengths were obtained from two-parameter least-squares fits of expression (5) to the excitation and the emission data, respectively.
  • the top graph (a) of Fig. 5 provides experimentally determined plots of phase shift versus modulation frequency for a 0.5 ⁇ M concentration of DTTCI with the excitation characterization measurements of stage 130, the emission characterization measurements of stage 140, and the fluorescence characterization measurements of stage 150 shown in ascending order.
  • the bottom graph (b) of Fig. 5 provides plots of the same type and in the same order as top graph (a) for a l.O ⁇ M concentration of DTTCI.
  • the top graph (a) and the bottom graph (b) of Fig. 6 provide excitation, emission, and fluorescence characterization plots in the same order as the graphs of Fig.
  • Fig. 7 plots the lifetime calculation results of stage 160 for the two concentrations for DTTCI (hollow symbols) and ICG (solid symbols) as based on the measurements illustrated in Figs. 5 and 6. It should be understood that optical parameters of the samples were not known a priori, and lifetime determinations were made without a reference luminophore, being based instead on the phase-shift measurements shown. Results of the two-parameter fits of expression (5) to the phase-shift data are indicated by curves that join the data symbols obtained at the excitation and the emission wavelengths. The optical coefficients extracted for the samples containing the various concentrations of DTTCI or ICG are summarized in Table 1.
  • Fig. 8 provides relative modulation magnitude versus modulation frequency plots of fluorescence, emission, and excitation characterizations for the two DTTCI concentrations as illustrated by different curve-fitted symbols.
  • Fig. 9 provides relative modulation magnitude versus modulation frequency plots of fluorescence, emission, and excitation characterizations for the two ICG concentrations as illustrated by different curve-fitted symbols.
  • Fig. 10 provides the lifetime calculations for the two concentrations of DTTCI and ICG based on the measurements plotted in Figs. 8 and 9.

Abstract

L'invention concerne un système (20), comprenant des instruments (30) de source lumineuse qui permettent d'éclairer sélectivement un milieu de dispersion de la lumière doté d'un luminophore, et des instruments de détection (50) qui permettent de détecter une sortie lumineuse dispersée plusieurs fois à partir du milieu, en réponse à un éclairement par les instruments (30) de source lumineuse. Un processeur (70) est couplé, de manière fonctionnelle, aux instruments de détection (50) afin de déterminer une première caractérisation optique du milieu, à partir d'une sortie lumineuse dispersée plusieurs fois d'une première longueur d'onde de lumière d'éclairement, et une deuxième caractérisation optique du milieu, à partir d'une deuxième sortie de lumière dispersée plusieurs fois d'une deuxième longueur d'onde de lumière d'éclairement différente de la première. Le processeur (70) fonctionne de manière à calculer la durée de vie du luminophore, à partir des première et deuxième caractérisations, et d'une émission du luminophore dispersée plusieurs fois à partir du milieu en réponse à une excitation.
PCT/US1999/023709 1998-10-09 1999-10-08 Caracterisation de la luminescence dans un milieu de dispersion WO2000022414A1 (fr)

Priority Applications (5)

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AU11099/00A AU1109900A (en) 1998-10-09 1999-10-08 Characterization of luminescence in a scattering medium
EP99954855A EP1127262A4 (fr) 1998-10-09 1999-10-08 Caracterisation de la luminescence dans un milieu de dispersion
CA2346728A CA2346728C (fr) 1998-10-09 1999-10-08 Caracterisation de la luminescence dans un milieu de dispersion
US10/473,303 US7054002B1 (en) 1999-10-08 1999-10-08 Characterization of luminescence in a scattering medium
NO20011791A NO20011791L (no) 1998-10-09 2001-04-09 Karakterisering av luminescens i et spredningsmedium

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US10360998P 1998-10-09 1998-10-09
US60/103,609 1998-10-09

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WO (1) WO2000022414A1 (fr)

Cited By (6)

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Publication number Priority date Publication date Assignee Title
WO2002082055A2 (fr) * 2001-04-03 2002-10-17 The Texas A & M University System Caracterisation de particules en suspension a partir de mesures des migrations photoniques par domaine de frequence
US6930777B1 (en) 2001-04-03 2005-08-16 The Texas A&M University System Method for characterizing particles in suspension from frequency domain photon migration measurements
US7054002B1 (en) 1999-10-08 2006-05-30 The Texas A&M University System Characterization of luminescence in a scattering medium
US7328059B2 (en) 1996-08-23 2008-02-05 The Texas A & M University System Imaging of light scattering tissues with fluorescent contrast agents
US7599732B2 (en) 2003-06-20 2009-10-06 The Texas A&M University System Method and system for near-infrared fluorescence contrast-enhanced imaging with area illumination and area detection
US7865230B1 (en) 1997-02-07 2011-01-04 Texas A&M University System Method and system for detecting sentinel lymph nodes

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US5624847A (en) * 1991-05-03 1997-04-29 Joseph R. Lakowicz Method for optically measuring chemical analytes
US5736410A (en) * 1992-09-14 1998-04-07 Sri International Up-converting reporters for biological and other assays using laser excitation techniques
US5818583A (en) * 1996-11-08 1998-10-06 Purdue Research Foundation Particle analysis system and method

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US5624847A (en) * 1991-05-03 1997-04-29 Joseph R. Lakowicz Method for optically measuring chemical analytes
US5736410A (en) * 1992-09-14 1998-04-07 Sri International Up-converting reporters for biological and other assays using laser excitation techniques
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7328059B2 (en) 1996-08-23 2008-02-05 The Texas A & M University System Imaging of light scattering tissues with fluorescent contrast agents
US7865230B1 (en) 1997-02-07 2011-01-04 Texas A&M University System Method and system for detecting sentinel lymph nodes
US7054002B1 (en) 1999-10-08 2006-05-30 The Texas A&M University System Characterization of luminescence in a scattering medium
WO2002082055A2 (fr) * 2001-04-03 2002-10-17 The Texas A & M University System Caracterisation de particules en suspension a partir de mesures des migrations photoniques par domaine de frequence
WO2002082055A3 (fr) * 2001-04-03 2003-12-04 Texas A & M Univ Sys Caracterisation de particules en suspension a partir de mesures des migrations photoniques par domaine de frequence
JP2005509135A (ja) * 2001-04-03 2005-04-07 ザ、テクサス、エイ、アンド、エム、ユーニヴァーサティ、システィム 周波数領域光子伝播計測により懸濁系内の粒子を特徴付けする方法
US6930777B1 (en) 2001-04-03 2005-08-16 The Texas A&M University System Method for characterizing particles in suspension from frequency domain photon migration measurements
US7268873B2 (en) 2001-04-03 2007-09-11 The Texas A&M University System Method for characterising particles in suspension from frequency domain photon migration measurements
US7599732B2 (en) 2003-06-20 2009-10-06 The Texas A&M University System Method and system for near-infrared fluorescence contrast-enhanced imaging with area illumination and area detection

Also Published As

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AU1109900A (en) 2000-05-01
CA2346728A1 (fr) 2000-04-20
NO20011791L (no) 2001-06-08
EP1127262A4 (fr) 2002-09-25
EP1127262A1 (fr) 2001-08-29
NO20011791D0 (no) 2001-04-09
CA2346728C (fr) 2013-04-23

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