WO2001084124A2 - Methods and systems using field-based light scattering spectroscopy - Google Patents
Methods and systems using field-based light scattering spectroscopy Download PDFInfo
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- WO2001084124A2 WO2001084124A2 PCT/US2001/040612 US0140612W WO0184124A2 WO 2001084124 A2 WO2001084124 A2 WO 2001084124A2 US 0140612 W US0140612 W US 0140612W WO 0184124 A2 WO0184124 A2 WO 0184124A2
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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
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/47—Scattering, i.e. diffuse reflection
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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
- G01N21/17—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
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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
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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
- A61B5/0062—Arrangements for scanning
- A61B5/0066—Optical coherence imaging
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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
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/41—Refractivity; Phase-affecting properties, e.g. optical path length
- G01N21/45—Refractivity; Phase-affecting properties, e.g. optical path length using interferometric methods; using Schlieren methods
Definitions
- Optical imaging techniques have been shown to be useful diagnostic tools for a number of biomedical applications.
- the structure of biological tissue is being studied using the interaction of the tissue with light.
- LSS light scattering spectroscopy
- the size and index of refraction of scatterers comparable in size to the wavelength of light are profiled by measuring the periodicity of the spectra of reflected light.
- sensitivity to variations in the size and index of refraction of cell nuclei can give indications of abnormal cell activity. For example, pre-cancerous epithelial cells exhibit nuclear enlargement.
- obtaining spectroscopic information from tissue in vivo is difficult as light scattered from surrounding tissues often obscures the desired optical signal.
- LSS light scattering spectroscopy
- OCT Optical Coherence Tomography
- OCT uses low- coherence interferometry to produce a three-dimensional image of optical scattering from internal tissue microstructures in a way that is analogous to ultrasonic pulse- echo imaging.
- OCT has longitudinal and lateral spatial resolutions of a few micrometers and can detect reflected signals as small as about 10 "10 of the incident optical power.
- tissue is placed in a Michelson interferometer illuminated by a broadband light source.
- A-Scan A map of tissue reflectivity versus depth
- the envelope, or outline of the detector current may be recorded with high dynamic range by scanning the reference mirror at fixed velocity, while demodulating the detector current at the resulting Doppler frequency.
- Cross-sectional images of tissue backscatter (called B-Scans) are acquired by collecting sequential A-scans while scanning the probe beam across the tissue surface.
- the resulting two-dimensional datasets are plotted as gray-scale or false-color images.
- OCT does not provide a functional image as the resultant resolution does not provide an image of the cell nuclei, whose size, shape and other characteristics can reveal abnormal cell activity.
- the present invention relates to systems and methods employing field-based light scattering spectroscopy to provide three-dimensional images and for determining physical characteristics of biological tissue.
- the three-dimensional images provide information regarding the characteristics of cellular or other structures including, for example, size, shape, texture of the cell nuclei as well as cellular dynamics which can include motility and chaos.
- Field-based LSS systems incorporate spectroscopic information into a low coherence interferometer system. This field-based type of LSS can be differentiated from intensity-based LSS as it is able to isolate small regions of a biological sample for study, and yields information regarding the phase, as well as the amplitude, of the scattered field.
- field-based LSS measures phase of the wavefront, and wavefront uniformity, as well as providing a high degree of spatial resolution
- field-based LSS can measure the shape and spatial distribution of cell nuclei along with size and refractive index of the nuclei. To ascertain the shape of cell nuclei, measurements of the scattered light's amplitude wavefront, phase wavefront, and wavefront non-uniformity are combined. Spatial distribution of cell nuclei is ascertained by three dimensional profiling which allows for imaging of cell nuclei density over the probed region.
- the field-based LSS system includes an angular light scattering system which measures both the position and angular distribution of a light field.
- Angular distributions provide information regarding variations in phase front uniformity (coherence of the scattered light) as well as indicate size of the scatterer.
- This system can include a light source such as a laser and an optical system that provides spatially coherent light having first and second wavelength components that are aligned to illuminate a region of interest of the material or tissue under study.
- a reference beam having both first and second wavelengths is also directed through the optical system along an optical path having an adjustable path length.
- An actuator is used to vary the path length at a selected speed such that detection of the reference beam and the light returning from the material under examination can generate a heterodyne signal which is and analyzed to determine selected characteristics of the material.
- the field-based LSS system includes a dynamic light scattering system to measure dynamic properties of tissue structure such as cell nuclei within a small selected region by combining field-based LSS with temporal measurements. Motions of small objects can be detected by scattering of laser light through examination of its statistical properties such as, for example, autocorrelation function and power spectra.
- a preferred embodiment of the invention involves a system for fiber optic delivery and a collection of light from tissue to provide real time diagnostic measurements either in vitro or in vivo.
- a preferred method of using the system involves analysis of the collected data by storing the data in memory of computer and comparing the collected data with reference data previously stored in electronic memory or database.
- Figure 1 illustrates a schematic diagram of a system using field-based light scattering spectroscopy in accordance with the present invention.
- Figure 2 graphically illustrates a theoretical fit of the data points measured in accordance with the present invention.
- Figure 3 illustrates a schematic diagram of a preferred embodiment of an angular light scattering system in accordance with the present invention.
- Figures 4A and 4B graphically illustrate theoretical angular distributions in accordance with the present invention.
- Figures 5 A and 5B graphically illustrate path delay and intensity measurements related to the angular light scattering system in accordance with the present invention.
- Figure 6 illustrates a block diagram of a preferred embodiment of a dynamic light scattering spectroscopy system in accordance with the present invention.
- Figure 7 graphically illustrates the correlation measurement related to the dynamic light scattering spectroscopy system in accordance with the present invention.
- Figure 8 schematically illustrates a fiber optic system used in conjunction with the present inventions.
- a preferred embodiment of a field based LSS system 10 including a Michelson interferometer with two low-coherence light sources.
- a Coherent MTRA Ti:sapphire laser 12 operating in femtosecond mode (150 fs) produces 800 nm light.
- the measured coherence length is about 30 ⁇ m.
- a portion of this light is handled by converter 15 such that it is split off and up-converted to 400 nm by means of a CSK Optronics LBO second harmonic generation crystal.
- two or more separate light sources can be used to provide the two or more wavelengths used for particular measurements. The converted light is then recombined with the original beam.
- the combined beam 14 is then divided by a beam-splitter 16 into a probe beam 18 and a reference beam 20.
- a single beam can be used with a variable wavelength source.
- the probe beam 18 is focused onto the sample by means of a 12.7 mm focal length achromatic lens 22.
- the powers of the 400 and 800 nm components at the sample are 14 and 5.5 mW, respectively.
- the beam waists at the lens are 1.1 and 2.1 mm (FWHM), resulting in beam waists at the focal point of 5.9 and 6.2 ⁇ m, and the Rayleigh lengths of 270 and 150 ⁇ m, respectively.
- the probe region can be approximately represented as a cylinder. The calculated beam overlap within this region is 98%, thus insuring that the same particles within the material 26 are illuminated simultaneously at both wavelengths.
- the reference beam 20 is reflected from a mirror 24 moving along linear axis 27 at a constant speed, inducing Doppler shifts of 14.6 and 7.3 kHz at 400 and 800 nm, respectively. It is then recombined with the probe light backscattered from the material 26 and transmitted to a dichroic mirror 30, which separates the 400 and 800 nm components and delivers them to separate auto-balanced photoreceivers 32,34 (New Focus 2007).
- Apertures 36, 38 are positioned in front of the detectors to limit the amount of collected light. In this particular embodiment the apertures have a radius of 0.9 mm. The distance from the collection lens 22 to the detectors is about 50 cm.
- a portion of the reference beam is separated with a second beamsplitter 25 and sent to a second dichroic mirror 40.
- the two output components from the mirror are delivered to the reference ports 42, 44 of the photoreceivers. This serves to cancel power fluctuations at the two wavelengths.
- the heterodyne signal at each wavelength which results from the interference of the back-scattered probe beam and the appropriate Doppler-shifted reference beam, is detected by the photoreceiver.
- Each signal is measured using a Stanford Research 830 lock-in amplifier array 46, 48.
- the magnitude of the heterodyne signal is displayed on a display 50 or oscilloscope and/or recorded by a computer 52.
- a cuvette with a thin layer of clear gelatin about 3 mm thick is followed by a layer of polystyrene microspheres suspended in gelatin.
- the probe beam enters the sample through the first layer and is brought to a focus at the interface between the first and the second layers.
- the two layers have the same refractive index, which ensures that there is no reflective interface between them. Therefore, any heterodyne signal observed can be attributed solely to scattering from the microspheres.
- the polystyrene microspheres range from 0.53 to 6.09 ⁇ m and can be obtained from Bangs Laboratories, Polysciences hie. and Spherotech hie.
- the volume concentration of microspheres within the second layer is maintained at 1.3% for these samples.
- the refractive index ratio of the microspheres to the gelatin is measured independently to be 1.19+0.01. To account for attenuation and absorption of the probe beam in the first layer of gelatin, the results are normalized using heterodyne signals from a cuvette in which the second layer of gelatin is replaced by a mirror.
- the amplitude of the measured heterodyne signal depends primarily on three factors: the number of microspheres illuminated, their positions within the probe beam, and the amount of back scattering they produce. Errors that may arise from the first two factors include that they can potentially skew the measurement and mask the periodic structure of the light scattering spectrum. These contributions can be minimized or eliminated by aligning the 400 and 800 nm beam components and employing approximately equal beam waists at the focus, as described above. This ensures that the beam components at both wavelengths illuminate the same region of the sample, and thus the same microspheres. Therefore, by taking the ratio of the signals at the two wavelengths, any dependence on the number and positions of the microspheres are normalized out.
- heterodyne signal from the interference of the reflection from a group of scatterers with a reference beam can be expressed as:
- E r (p) the electric field of the reference beam incident on the detector
- p the spatial coordinate on the detector's surface
- E r (p) is the amplitude component of the signal electric field which is polarized in the same direction as the reference beam.
- ⁇ denotes the heterodyne frequency
- t represents time
- ⁇ ⁇ (p) the associated phase shift of
- ⁇ (p) denotes the amplitude of the scattered electric field associated with
- the reference field is represented as having a Gaussian transverse spatial variation across
- the quantity & is the spatial coherence factor. It measures the
- E/ , ,(p) is identical in profile to E r (p) . Note that E r and E ⁇ are simply the
- heterodyne measurement strongly favors the detection of singly scattered light over multiply scattered light, provided that the two are present in comparable amounts. Such is the case for light scattered near the surface of a turbid medium such as tissue.
- the measured heterodyne signal can be
- a( ⁇ ,D) is a
- N can be found by measuring the backscattered intensity at many wavelengths. More details regarding such measurements can be found in U.S. Application No. 08/948,734 filed on October 10, 1997. the entire contents of the referenced application being incorporated herein by reference. The same can be done in field-based LSS.
- N cannot be accurately determined.
- the beam components at the two wavelengths are well aligned, and thus illuminate the same focal area, the same scatterers are simultaneously illuminated at both wavelengths in each measurement.
- field-based LSS is also influenced by the wavelength-dependent spatial coherence factor, a ( ⁇ , D) . This indicates that the field-based LSS spectrum differs from the intensity-based. LSS spectrum.
- the ensemble averaged quantity a ( ⁇ , D) ⁇ ( ⁇ , D) can be calculated from
- Mie scattering theory for spherical objects First express the contribution to the heterodyne signal from a single scatterer at a displacement d t from the focal point. This is given by:
- the predictions for R for microspheres ranging from 0.1 to 7.0 ⁇ m in diameter can be determined.
- the result is then averaged over a 5% variation in diameter, to account for the distribution of microsphere sizes in a given sample. This distribution is consistent with the size distribution given by the manufacturers.
- Measured data are plotted in Figure 2. Each data point consists of 30 or 45 measurements taken at various sample positions. Averaging over scatterer position is determined by taking the root mean square of the measured heterodyne signal at each wavelength. The ration of the results, R, is then calculated and plotted.
- the solid line in the plot is the theoretical fit.
- the fit was done using the procedure outlined above, hi the fitting procedure, the aperture size of the detectors were free parameters.
- the best fit requires an aperture size of 0.7 mm in radius, instead of 0.9 mm, for the 400 nm measurements. This can be accounted for as a possible misalignment of the incoming beam at the detector, which can result in a smaller effective detection area.
- field-based LSS determines the size and relative refractive index of scatterers by measuring variations in scattering across the spectrum
- field-based LSS differs from intensity-based LSS in two important ways. First, it is sensitive to phase front variations in the scattered wave, as well as to the intensity variations of the back-scattered light. Second, it permits greater localization of the region to be measured.
- Single particle light scattering is characterized by the scattering amplitude, S( ⁇ , D, ⁇ , ⁇ , ) , which is a complex function with a phase that varies with angular coordinates. This phase is not measured in intensity-based LSS. In contrast, field- based LSS is very sensitive to variations in phase. To illustrate this, consider a measurement with a plane wave incident field. The average scattering intensity from a single scatterer measured at the detector can then be expressed as:
- phase variations become important in field-based LSS, and the measured signal is proportional to the degree of phase-front uniformity of the scattered light.
- the phase front uniformity degrades as a function of f— 1 , and so for a scatterer of a given size, there is more
- the theory employs an ensemble-averaged spatial coherence factor, d( ⁇ ,D) , to quantify the degree of phase front uniformity. Note that ⁇ ( ⁇ , D) is fundamentally related to f( ⁇ ,D, ⁇ , ⁇ ) .
- Eq. (12) is applicable only in situations where both the reference and incident fields are uniform, and are appropriately modified for non-uniform input fields.
- the scattering cross-section, ⁇ ( ⁇ , D) , and the anisotropy factor, g( ⁇ ,D) are often used to characterize the scattering process.
- specification of the scattering process in field-based light scattering requires a third parameter derived from S( ⁇ ,D, ⁇ , ⁇ ) the spatial coherence factor a( ⁇ ,D) .
- field-based LSS has the advantage of being able to localize a much smaller region for study.
- This localization can be achieved with low-coherence interferometric techniques, as exemplified by OCT.
- Spatial localization is limited by the coherence length, and a typical femtosecond laser beam can localize a region for study on the order of tens of microns.
- This opens the possibility of selectively probing each individual scatterer, such as a single cell nucleus, even when it is surrounded by other scatterers. The prospect of probing one scatterer at a time also simplifies the computations. For a single scatterer, the cross-tenns in Eq.
- FIG. 3 illustrates another preferred embodiment 60 of the invention in which a source of light 82, a beamsplitter 80 which splits the light along two paths, a first path extends through lens 74 onto the tissue or object being measured and the second path is directed through lens 66 reflects off mirror 64 and is redirected through the beamsplitter 80 along with light returning from the object being measured through lens 78 to detector system 76.
- both the lens 66 and mirror 64 are displaced over a distance 70 during the measurement. Additionally, the lens 66 can be displaced in a second direction 68 to alter the direction of propagation.
- the position of the reference field in the detector plan remains fixed.
- Figures 4A and 4B graphically illustrate the angular dependence of measurements made using the present invention.
- Figures 5A and 5B illustrate path delay and intensity measurement of a monolayer. The angular resolution of this measurement is about 0.45 mrad, and the longitudinal resolution is about 11.6 ⁇ m.
- Figure 6 schematically illustrates a process in which measurements employing autoconelation methods provide information and images of changes in cellular or subcellular structures in tissue.
- Figure 7 graphically illustrates the results of measurements of conelation in the intensity of light from a He-Ne laser scattered by a dilute suspension of beads. The autoconelation time of 0.51 msec is consistent with multiple scattering of the light by 0.22 micron beads.
- a fiber optic system 200 for light delivery and/or collection in conjunction with the light scattering spectroscopic systems and methods of the invention described previously.
- a light source provides a beam 202 that includes at least two wavelengths ⁇ _, ⁇ 2 which are coupled to the proximal end of optical fiber 204.
- a beam splitter 206 incorporated into the fiber optic system delivers light components through fibers 208 and 210, and through lenses 216 and 214, respectively.
- a first light component is reflected by moving minor 220 traveling in direction 220, and returns through fibers 210 and 212.
- a second light component is directed onto tissue 218, and light scattered by the tissue is returned through fibers 208 and 212.
- Dichroic minor 230 separates the two wavelength ⁇ 1 and ⁇ _ which are detected by detectors 240 and 242, respectively.
- the heterodyne detection systems 250 and 252 are used to process the detected systems as described previously in connection with Figure 1.
- the systems described herein can be used in conjunction with standard endoscopics to provide diagnostic information retrieved from lumens or tissue within the human body in vivo. While this invention has been particularly shown and described with references to prefened embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
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Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
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AU59807/01A AU5980701A (en) | 2000-04-28 | 2001-04-27 | Methods and systems using field-based light scattering spectroscopy |
EP01933374A EP1277040A2 (en) | 2000-04-28 | 2001-04-27 | Methods and systems using field-based light scattering spectroscopy |
KR1020017016801A KR20020027377A (en) | 2000-04-28 | 2001-04-27 | Methods and systems using field-based light scattering spectroscopy |
JP2001581098A JP5134177B2 (en) | 2000-04-28 | 2001-04-27 | Systems using light-scattering spectroscopy based on electric fields |
CA002377724A CA2377724A1 (en) | 2000-04-28 | 2001-04-27 | Methods and systems using field-based light scattering spectroscopy |
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US9528817B2 (en) | 2001-12-18 | 2016-12-27 | Massachusetts Institute Of Technology | Systems and methods for phase measurements |
Also Published As
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EP1277040A2 (en) | 2003-01-22 |
CN1279345C (en) | 2006-10-11 |
US6847456B2 (en) | 2005-01-25 |
WO2001084124A3 (en) | 2002-06-20 |
US20020101593A1 (en) | 2002-08-01 |
JP5134177B2 (en) | 2013-01-30 |
KR20020027377A (en) | 2002-04-13 |
AU5980701A (en) | 2001-11-12 |
CA2377724A1 (en) | 2001-11-08 |
CN1451094A (en) | 2003-10-22 |
JP2003532112A (en) | 2003-10-28 |
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