JP4751271B2 - Photoacoustic analysis method and photoacoustic analysis apparatus for measuring the concentration of an analyte in a specimen tissue - Google Patents

Description

  The present invention is a method and apparatus for non-invasive light measurement for measuring the concentration of an analyte in a subject tissue using photoacoustic interaction. More particularly, the present invention is a method that compensates for the effects of tissue properties on the generation, propagation and detection of photoacoustic signals, thus leading to quantitative analysis of analytes in the tissue.

  Photoacoustic measurement is an alternative detection technique for the interaction of near-infrared light with tissue. Photoacoustic spectroscopy is used to detect weak absorbance of a liquid or gas (see Non-Patent Document 1). The tissue is excited by a pulse of near-infrared laser light at a wavelength that is absorbed by molecules having an absorption band in the region of laser excitation. Subsequent optical absorption causes minute local heating in the medium. The rise in temperature causes rapid thermal expansion, which can be detected by an ultrasonic transducer such as a hydrophone, piezoelectric element, or polyvinylidene fluoride (PVDF) film placed on the surface of the measurement cell or on the skin surface. Generate a wave.

  The conversion of light pulses into pressure (ultrasound) pulses is effected by the photoacoustic effect. A study of the spectral distribution of the intensity of the measured ultrasonic pulse as a function of the wavelength of light is photoacoustic spectroscopy. Oraevsky et al discussed the theory of photoacoustic spectroscopy in an academic paper (Non-Patent Document 2). Their gist on this technique showed that the pressure generated by irradiating the tissue is related to the extinction coefficient of the medium as follows:

ΔP = [(βv ² ) / C _p ] μ _a . E ₀ (1)
This equation can be expressed as three terms.
Bulk medium thermal response term (absorption coefficient) = [(βv ² ) / C _p ]
Absorber characteristic term = μ _a expressed in cm ⁻¹
Source energy fluence term = E ₀ expressed in joules per cm ²
However,
E ₀ is the laser fluence expressed in joules per cm ² ,
μ _a is the extinction coefficient of the absorbing species in the medium,
β is the thermal expansion coefficient of the medium or tissue, and (° K ⁻¹ )
v is the speed of sound in the medium or tissue,
C _p is the specific heat of the medium under a constant pressure.

  The photoacoustic signal measured as the pulse amplitude by the ultrasonic transducer after the pressure wave has traversed the medium is expressed as follows.

PA = ΔP · RTDΛS (2)
However,
PA is the ultrasonic pulse amplitude created as a result of photoacoustic interaction;
ΔP is the pressure change in the medium as a result of the photoacoustic interaction;
R is a stress relaxation factor that depends on the laser pulse width,
T is the acoustic transmission through the interface,
D is the acoustic diffraction pattern in the medium or tissue,
Λ is the attenuation of the sound wave in the medium or tissue = e ^−αd , α is the acoustic attenuation coefficient of the absorber in the medium or tissue, and d is the distance traveled by the sound pulse,
S is the response of the ultrasonic detector.

Substituting from equation (1) to equation (2) yields:
PA = RTDΛS. [(Βv ² ) / C _p ] μ _a . E ₀ (3)
Conventional studies correlate the value of (PA / E ₀ ) with the analyte concentration (ie, its extinction coefficient μ _a ), which indicates that the total amplitude of this measured ultrasound pulse is It is assumed that it is generated as a result of light absorption. All of the photoacoustic signal amplitude change does not mean that contributed by the absorption coefficient mu _a solute was added to the medium (contribution). The addition of a solute to a medium actually affects the internal thermoelastic properties of the medium and the propagation of ultrasonic waves in the medium. Thus, in the case of non-invasive measurement of glucose, the generated ultrasonic pulse amplitude depends on the extinction coefficient μ _a of the analyte in the medium, and also depends on another parameter in equation (3).

The amplitude of the measured ultrasonic pulse obtained from the photoacoustic in the medium is the analyte for three parameters: the thermal expansion coefficient β of the medium, the speed of sound v in the medium, and the specific heat C _p of the medium at a constant pressure. Depends on the concentration effect. In a given solution or tissue, beside the sound velocity v with increasing glucose concentration increases, C _p of the solution or tissue decreased β is affected. When the laser pulse excites the solution at the glucose absorption wavelength, the resulting change in the amplitude of the ultrasonic pulse is due to changes in μ _a , v, C _p and β. The synergistic effect of these three parameters increases the photoacoustic signal as a function of concentration, which slightly complicates the relationship between concentration and photoacoustic signal.

The values of v, C _p and β depend on the surface characteristics of the subject site such as tissue packing (packing), tissue dimensions (such as finger or earlobe size), presence of calluses and surface irregularities. To do. Since v, C _p and β all vary with temperature and pressure, the thermoelastic properties of the tissue depend on the pressure applied thereto and the temperature.

  The measured signal depends on factors that affect the propagation of the ultrasonic pulse after it is generated, and also depends on the ability of the detector. Thus, the measured signals are the stress relaxation factor (R), the acoustic voltage (T) through the interface, the acoustic diffraction pattern in the tissue (D), the acoustic attenuation in the tissue (Λ), and the ultrasonic detection Depends on the response (S) of the vessel. The parameters R, T and S depend on the device settings. Parameters D and L vary from person to person and depend on the solute concentration. These three factors vary from person to person and vary from experiment to experiment, making direct measurement of the analyte concentration in the tissue impossible and only monitoring the relative change in analyte concentration.

  The amplitude of the transducer signal is a composite of the signal component related to the concentration of the absorbing species and the signal component related to the influence of the medium (tissue) on the generation and propagation of ultrasound from the generation point to the detector. .

  Pulsed photoacoustic (PA) techniques are described in A.D. A. Kalabtov and A. A. It is reviewed in a book by A. A. Karabutov, A. A. Oraevsky (Non-Patent Document 3). The PA detector collects all the signals generated in all the sampled volumes in the excited tissue and detects the signals generated at wavelengths longer than the operating spectral range of the silicon or gallium arsenide detector. Measurement is somewhat superior in detection sensitivity over near infrared detection methods. Therefore, it is important to use the photoacoustic effect for measuring the analyte in the tissue. However, attempts to quantify analytes in tissue are hampered by the inability to separate the effects of molecular absorption from the effects of bulk thermoelasticity on the generation and propagation of detected ultrasound signals. Therefore, a method that allows to separate or compensate for the effects of tissue internal properties on the generation and propagation of detected ultrasound pulse amplitudes is a method of analyzing analytes in tissue using photoacoustic spectroscopy. It is critical for non-invasive measurements.

  The Rosecenwaig US patent (US Pat. No. 5,697,075) proposed the use of photoacoustic measurements for the analysis of substances, but does not disclose its use for the measurement of analytes in human tissue. Not.

  Dowling is a published European patent application (Patent Document 2) that proposes to use photoacoustic spectroscopy for blood analysis measurements of glucose, etc., but calculates the glucose concentration in the tissue. There is no discussion or suggestion of a method for compensating for the influence of the internal characteristics of the subject site on ultrasonic propagation.

  Patel and Tam, US Pat. No. 6,037,099, used nanosecond laser pulses to excite the photoacoustic effect in solids for the measurement of analytes in human tissue. Is not disclosed.

  Caro's US patents (Patent Documents 4 and 5) have proposed using photoacoustic measurements in combination with light absorption to measure blood components in vivo. Caro has not proposed any way to compensate for the effects of internal properties on the generation and propagation of ultrasound within the tissue in order to calculate the concentration of glucose.

  McKenzie et al. (Patent Document 6) and US Patent (Patent Document 7) described a method for measuring a signal in the direction of high acoustic energy. Mackenzie et al. Do not disclose or teach any method to compensate for the effects of tissue thermoelasticity on the generation and propagation of ultrasound in tissue to calculate the concentration of glucose in the tissue.

  The Chou U.S. Pat. No. 6,057,836 describes a method for detecting a photoacoustic signal using a probe, which includes a measurement cell, a reference cell, a window and a differential microphone. The excitation wavelengths are in the range of 1580-1850 nm and 2050-2340 nm. Chow does not disclose or teach any method to compensate for the effects of tissue thermoelasticity on the generation and propagation of ultrasound within the tissue to calculate the concentration of glucose in the tissue.

  Olaevsky and Karabtov (US Pat. No. 6,057,097) disclose a pulsed photoacoustic method where pulse shape analysis gives information about the scattering coefficient, which is later correlated with glucose. Olaevsky et al. Do not disclose or teach any method for compensating for the effects of tissue thermoelasticity on the generation and propagation of ultrasound in tissue to calculate the concentration of glucose in the tissue.

  Thus, conventional photoacoustic spectroscopy has failed to provide a viable method or practical device for non-invasive measurement of glucose in human tissue. This is because conventional methods neglected the contribution of tissue internal thermoelastic properties to the generation and propagation of ultrasound in human tissue. Conventional methods compensate for this influence of the contribution of the tissue's internal thermoelastic properties on the generation and propagation of ultrasound in human tissue in the measured signal, and thus on the measured glucose concentration in human tissue. Is not discussed.

Epstein et al., US Pat. No. 6,057,049, describes a human for monitoring the concentration of an analyte with continuous laser pulses and the use of a thermally conductive light absorbing dye in contact with the skin. A method for microporation of the skin has been disclosed. The heat generated by the interaction between the laser and the intensity absorbing dye corroded the stratum corneum and opened a hole in the skin. Next, interstitial fluid was collected from the drilled holes. The method of Patent Document 11 includes sampling of a body fluid and does not describe a non-invasive photoacoustic measurement method. Jacques et al., US Pat. No. 6,057,086, describes a controlled removal of the human stratum corneum with a pulsed laser to collect blood for chemical analysis. The use of a pulsed laser by Epstein and Jack is not intended to generate a photoacoustic wave in tissue to measure glucose in a non-invasive manner without destroying the stratum corneum, but to extract a fluid sample from human tissue It was a thing.
US Pat. No. 3,948,345 European Patent Application No. 0822234A1 US Pat. No. 4,303,343 US Pat. No. 5,348,002 US Pat. No. 5,348,003 International Publication No. 98/38904 Pamphlet US Pat. No. 6,403,944 US Pat. No. 5,941,821 US Pat. No. 6,405,069 US Pat. No. 6,846,288 US Pat. No. 5,885,211 U.S. Pat. No. 4,775,361 A. C. General Review by A. C. Tam, “Application of photoacoustic sensing techniques”, Review of Modern Physics (Reviews of Modern Physics), Vol. 58, No. 2, 1986, p3831-4 Orevsky et al., “Measurement of tissue optical properties by time-resolved detection-induced in- duced-in- duced-in- duced-in- duced-in- duced-in- duced-induced-optics-induced-induced-optics-induced-optical-induced-optics-induced-induced-optical-induced-optics-induced-optical-induced-optics-induced-optics-induced-optical-induced optics), 1997, volume 36, p. 402-415 A. A. Kalabtov and A. A. Olaevsky (A. A. Karabutov, A. A. Oraevsky), “Time-resolved detection of opto-stressed measures of measurement”. Handbook of Optical Biomedical Diagnostics, Valery V. Tuchin, Ed. , SPIE Press, Billingham, WA, USA, 2002, Chapter 10, p585-646.

  An object of the present invention is to provide a photoacoustic analysis method that compensates for the influence of bulk tissue characteristics on non-invasive measurement of an analyte concentration in a target tissue with respect to an ultrasonic pulse generated and propagated in the target tissue in the subject, and Providing the device.

In one aspect of the present invention, the photoacoustic analysis method includes signal amplitudes from the effects of variations in tissue properties on the signal and from the effects of the target analyte or other tissue components on the internal thermoelastic properties of the tissue. Results in a normalized signal that can be correlated with the concentration of the analyte without a contribution to. The method also corrects tissue and transducer effects on ultrasound generation, propagation, and propagation and detection.
Another aspect of the present invention is a method for compensating for the effect of variable tissue properties on the generation and propagation of ultrasound signals induced by absorption of short-time laser pulses by tissue components.
According to the method of the present invention, the photoacoustic signal is generated and detected under two different conditions with respect to the subject site.
a) A natural light absorber in the region of the subject such as hemoglobin, water, fat and glucose is excited at a wavelength specific to this naturally occurring absorption molecule, and an ultrasonic pulse obtained from this absorption event is detected.
b) An external absorber having a known absorbance that is bound to the tissue of the subject site is excited at the same temperature, pressure, and wavelength as used in step (a), and in the presence of the external absorber. The ultrasonic pulse resulting from this absorption event at is detected.
Using these two signals and the optical properties of the external absorber, the extinction coefficient and thus the concentration of the analyte of interest in the human tissue is determined. The term external absorber refers to a substance added to a medium or another substance that is strongly bound to a biological system. It absorbs light with a much higher extinction coefficient than the molecule of interest, and its absorption does not change over time, as does the method of extinction of the control analyte.
The excitation of the photoacoustic (PA) signal within the subject site in the natural state and the measurement of the generated and propagated ultrasound signal is near-red in hemoglobin, water, glucose or other desired analytes. This is accomplished by irradiating the subject site with laser pulses at one or more wavelengths within the outer spectral absorption band. Ultrasound signal corresponds to the photoacoustic signal by the light absorption by the natural absorption of the tissue (mu _a term). It also has a contribution due to the influence on the pulse generation of the internal thermoelastic properties of the tissue due to changes in β and C _p . The signal also has contributions from solute effects, interface effects and ultrasonic detector responses on the mechanical and acoustic propagation properties (v, D and Λ) of tissue.

One aspect of the invention is a method for the measurement of an analyte such as glucose in human tissue comprising the steps of: a. Generating an ultrasonic pulse by irradiating a subject site with a near-infrared short-time laser pulse in human tissue; b. Detecting a first signal obtained as an ultrasonic pulse associated with each excitation wavelength; c. A light absorbing material is added to the optical path between the laser source outside the tissue and the tissue so that an ultrasonic pulse is generated in the light absorbing material and propagates into the tissue to the ultrasonic detector. Generating ultrasonic pulses in the same tissue using the same laser wavelength and energy; d. Detecting a second signal obtained in the presence of the external light-absorbing substance as an ultrasonic wave generated at each wavelength; e. Calculating a ratio of the intensity of the pulse generated in step (b) to the intensity of the pulse generated in step (d) at each irradiation wavelength; f. Calculating the extinction coefficient of the analyte in the tissue from the ratio of the signal intensity determined in step (e) and the predetermined optical properties of the external light-absorbing substance; g. Calculating the concentration of the analyte from the extinction coefficient calculated in step (f) and the molar extinction coefficient of the analyte at the photoacoustic excitation wavelength.
In another aspect of the present invention, in the photoacoustic analysis method for compensating for the influence of the internal characteristics of the tissue on the signal obtained by measuring the analyte in the target tissue of the subject site, a laser is applied to the subject site. (A) generating an ultrasonic pulse in the target tissue by irradiating a near-infrared laser pulse from a source, and detecting the ultrasonic pulse using an ultrasonic detector disposed in the vicinity of the subject site Generating a first signal in step (b), and generating the first pulse in the light-absorbing material to propagate through the tissue and be detected by the ultrasonic detector. During the period in which the light-absorbing substance is disposed between the specimen site and the step (b), a laser pulse is generated in the target tissue using a laser pulse having the same laser wavelength and energy. (C), a step (d) of detecting an ultrasonic pulse generated in the light-absorbing substance and generating a second signal, a first signal generated in the step (b), and the step (d) (E) calculating the second signal and the ratio generated for each laser wavelength, the extinction coefficient of the analyte, the ratio calculated in step (e), and a predetermined value of the light-absorbing substance. Step (f) calculated from the optical characteristics, and the concentration of the analyte is calculated from the absorption coefficient calculated in the step (f), and the molar absorption coefficient of the analyte at the photoacoustic excitation wavelength is calculated. Step (g).

  According to the present invention, in the photoacoustic measurement, the influence of the bulk tissue characteristic on the noninvasive measurement of the analyte concentration in the subject tissue can be compensated for the amplitude of the ultrasonic pulse.

  Embodiments of the present invention provide for the amplitude of an ultrasound signal induced by photoacoustic effects in tissue of the influence of the internal characteristics of the subject site on the generation and propagation of ultrasound pulses in human tissue. Provides a means to compensate for the effects on. The resulting normalized signal is used to measure the concentration of the analyte in human tissue.

The compensation method of an embodiment of the present invention allows the analyte concentration to be measured without any contribution to the signal amplitude from the effect on the tissue internal properties, specifically β, C _p , D and Λ. The result is a photoacoustic signal that can be correlated with the analyte concentration. The method further corrects tissue and transducer effects on the propagation and detection of ultrasound pulses (R, D, Λ, T and S).

  Embodiments of the present invention are methods for compensating for the effects of variable tissue internal properties on ultrasound signals generated by photoacoustic effects and propagating through a subject site. The method allows non-invasive measurement of analytes in human tissue.

According to the method of the embodiment of the present invention, the photoacoustic signal is generated and detected under two different conditions with respect to the subject site.
a) Spontaneous light absorbers such as hemoglobin, water, fat and glucose are excited at a wavelength specific to the spontaneously absorbing molecule, and an ultrasonic pulse obtained from the light absorption event is detected.
b) A light-absorbing substance external to the human body that is bound to the tissue of the subject site and has a known optical density is excited at the same temperature and pressure and the same wavelength as used in the previous step, and the external light-absorbing material The ultrasonic pulse resulting from this absorption event occurring in the presence of is detected.

  Measure the extinction coefficient of these analytes in human tissue using two signals from the natural light absorber in the human body and from the light-absorbing material outside the specimen site, together with the optical properties of the external light absorber To do. The concentration of the analyte is calculated by dividing the absorption coefficient by the extinction coefficient of the analyte at the photoacoustic excitation wavelength. A flowchart illustrating the method of an embodiment of the present invention is shown in FIG.

The excitation of the photoacoustic signal in the human body and the measurement of the ultrasound signal generated naturally at the specimen site is within the near-infrared spectral absorption band of hemoglobin, water, glucose or other desired analyte. This is accomplished by irradiating the subject site with a laser pulse at a defined wavelength. The ultrasound signal corresponds to a photoacoustic signal generated as a result of light absorption by the natural light absorber in the tissue. The signal further receives contributions from the influence of tissue internal properties on the generation and propagation of ultrasound pulses. In addition, the signal receives contributions from the analyte's influence on the thermoelastic properties in the tissue, such as β, C _p , v, D and Λ.

The intensity of the measured ultrasonic pulse obtained from excitation of the naturally absorbed molecules found in the tissue comprises the following steps.
a. Naturally occurring molecules absorb light from laser pulses.
b. Pressure waves (ultrasonic pulses) are generated in the tissue and propagate through the tissue mass to the detector.
c. The internal properties of the tissue affect the generation and propagation of the resulting ultrasonic pulse.
d. The detector converts the ultrasonic pulse into an electrical signal whose intensity depends on the collection efficiency and the sensitivity of the transducer.
The intensity of the measured ultrasonic pulse obtained from excitation of the light-absorbing substance that is outside the subject site and brought into contact with the tissue comprises the following steps.
a. The external extinction material bound to the tissue absorbs most of the light from the laser pulse due to its high absorbance value (optical density).
b. A pressure wave (ultrasonic pulse) is generated in the external absorption layer at the interface with the tissue and propagates through the tissue mass to the detector.
c. The internal properties of the tissue affect the generation and propagation of the resulting ultrasonic pulse.
d. The detector converts the ultrasonic pulse into a voltage signal whose intensity depends on the collection efficiency and the sensitivity of the transducer.

  The light-absorbing substance outside the subject site is a substance having a known high optical density that exceeds 1.0, for example, at all wavelengths used for excitation (that is, it absorbs light intensity exceeding 90%). is there. When the laser pulse acts on the external light absorber coupled to the subject site, an ultrasonic pulse is generated and propagates to the tissue at the subject site. The ultrasonic pulse resulting from the photoacoustic excitation of the external absorber bound to the tissue is then measured. Calculate the concentration of analytes such as glucose, hemoglobin, water, and lipids in human tissue using the signal from the excitation of spontaneously absorbing molecules in the tissue and the signal in the presence of the natural absorber .

  There are several methods used in ultrasound imaging to minimize ultrasound reflection and ensure that the correct coupling of the detector to the skin is used in the method of the present embodiment. Thus, an ultrasonic coupling gel can be applied to achieve a highly efficient coupling between the skin and the detector. It can also be applied between the skin and an external absorber to achieve a highly efficient bond.

  The illumination of the sample by the light source and the generation of the photoacoustic signal can be achieved by a device similar to a conventional instrument. An example of a device configuration for generating and detecting a photoacoustic signal is shown in FIG. 2A.

  The device (10) comprises a light source module (11), a body part interface module (25), and a detection electronics module (40). The light source module (11) includes a power source (12) and laser sources (15-18). Quartz optical fibers (20-23) are connected to the laser sources (15-18), respectively. These fibers (20-23) are connected to the end of a lens housing (26) that contains a condenser lens (27) that focuses the light onto the sample (28). A piezoelectric detector (32) is mounted on the holder (34) on the opposite side of the lens in alignment with the beam in transmission arrangement. In the case of a reflective arrangement, the detector (32) and the collection optics will be on the same side as the sample. In the vertical arrangement, the detector (32) and the collection optics are at right angles to each other. Other arrangements at other angles can also be designed. It is also possible to change the position of the detector to a vertical arrangement (perpendicular to the illumination beam). The detector (32) is kept in intimate contact with the sample or subject site via the mechanism (35) by adding known weights or by controlling the movement of the tissue under known weights. .

  The detection electronics / module (40) includes a disconnect connector (41), an amplifier (42), a digital storage scope (43), and a control PC (44). The PC (44) is connected to the scope (43) by a signal line (46), and is connected to the power source (12) of the light source module 11 by a signal line (48). The power source (12) is also connected to the scope (43) by a signal line (50).

In this embodiment, an ultrasonic transducer described in International Publication No. 2004 / 1042382A1 pamphlet is used as the detector (32). This transducer is used in reflection mode, in which case a back-propagated ultrasonic pulse is detected. The piezoelectric device in the form of a single crystal containing lead titanate is represented by the general formula Pb [(B1, B2) _1-x Ti _x ] O ₃ and has a fraction x = 0.5 to 0.55, and B1 Represents an element selected from Zn, Mg, Ni, Sc, In and Yb, and B2 represents one element selected from niobium, Nb, tantalum and Ta. These crystalline transducers known as PZNT transducers can pass near infrared light and detect ultrasound. Therefore, the PZNT crystal is used as a window for light excitation and as a detector for ultrasonic waves generated by light pulses.

  In another embodiment, PZNT crystals are coated with a polymer film having acoustic propagation properties that match those of human skin.

  Another device configuration example is shown in FIG. 2B. The instrument comprises a light source module (100), a body part interface module (110), and a detection electronics module (120). The light source module (100) includes a power source (103) and a light source (102) such as a lamp. A quartz optical fiber (104) is connected to the light source (102). The fiber (104) is connected to the end of a lens housing (111) containing a condenser lens (112) that focuses the light onto the sample (113). A piezoelectric detector (114) is mounted on the holder (115) in line with the beam in transmission arrangement on the opposite side of the lens. In the reflective arrangement, the detector (114) and the collection optics will be on the same side as the sample. In the vertical arrangement, the detector (114) and the collection optics are at right angles to each other. Other arrangements at other angles can also be designed. It is also possible to change the position of the detector to a vertical arrangement (perpendicular to the illumination beam). The detector (114) is kept in intimate contact with the sample or subject site via the mechanism (116) by adding known weights or by controlling tissue movement under known weights. .

  The detection electronic circuit module (120) includes a disconnect connector (117), an amplifier (121), a digital storage scope (123), and a control PC (125). The PC is connected to the scope (123) by the signal line (124), and is connected to the power source (103) of the light source module (100) by the signal line (126). The power source (103) is also connected to the scope (123) by a signal line (127).

  For in vivo experiments, the sample is placed in a quartz cell, which contacts one side to a PZNT ultrasound detector coated with a 1 μm gold layer. FIG. 3 shows the photoacoustic signal as a function of concentration of glucose added to the medium and irradiated at 905 nm, 1459 nm, 1550 nm and 1649 nm. Three plots were obtained from plots of signals at 1459 nm, 1550 nm and 1649 nm for glucose. 1459 nm is the absorption wavelength of water, and 1550 nm and 1649 nm are the wavelengths of glucose. When the aqueous glucose solution was excited at 905 nm or less, there was no dependency that the photoacoustic signal could be detected on the glucose concentration.

FIG. 3 shows that the PA signal (photoacoustic signal) at 905 nm has zero slope and that there was no PA signal at the concentration (0-20 g / dL) used in the experiment. . There was a PA signal that could fit as a line at 1459 nm, PA (mv) at 1459 nm = 11.366 [glucose g / dL] +7.7661, R ² = 0.9757. When excited at 1550 nm and 1649 nm, there is a weaker PA signal that can fit as a straight line, PA (mv) at 1550 nm = 5.1247 [glucose g / dL] +5.0825, R ² = 0.9569 And PA (mv) at 1649 nm = 5.2031 [glucose g / dL] + 9.3373, R ² = 0.9224. The linear slope of the PA signal versus glucose concentration at 1459 nm was twice the linear slope of the glucose absorbance wavelength at 1550 nm and 1649 nm.

  The behavior of the photoacoustic signal excited at the water absorption wavelength of 1459 nm was unexpected. The pulse amplitude increased with increasing glucose concentration. It has been shown that the absorption process at 1459 nm, followed by the generation of ultrasound pulses, increases in intensity as the glucose concentration increases. If the photoacoustic signal is due to absorption by glucose molecules, the ultrasonic pulse amplitude and infrared absorption spectrum of the solution should match, or at least change in parallel with the change in the photoacoustic signal.

  FIG. 4 shows an infrared absorption spectrum (performed on a Bruker FT-IR spectrometer in a 1 mm cell) of a glucose solution. Light absorption at 1459 nm decreases with increasing glucose concentration, which is in stark contrast to the PA signal behavior as a function of glucose concentration shown in FIG. The decrease in glucose absorbance in the water absorption band is well known in the art and is due to the replacement of water molecules with glucose molecules.

There is a difference between the photoacoustic signal excited at 1459 nm and the near-infrared absorption signal of the aqueous glucose solution around 1459 nm. In these graphs, it is clear that the photoacoustic signal and the water absorption wavelength of the aqueous glucose solution depend on the glucose concentration. One interpretation of that dependence is that water absorbs 1459 nm photons and a photoacoustic signal is generated, which is then modulated by the medium, particularly due to the effect of glucose concentration on the internal properties of the solution. . The internal properties of these solutions include the values of β, v, C _p , D and Λ in equation (3) as it propagates through the cell to the detector. Thus, the photoacoustic spectroscopy of the prior art neglects the role that the influence of the analyte concentration such as glucose concentration on the internal thermoelastic characteristics of the medium plays on the intensity of the photoacoustic signal.

  Another example of the effect of added solute concentration, such as glucose, on the internal thermoelastic properties of the medium and thus the amplitude of the photoacoustic signal is shown in FIG. The glucose concentration was adjusted with a 0.9% sodium chloride solution containing a fixed amount of near infrared absorbing dye having an optical density of 2 units per cm at 905 nm. Four linear response plots were obtained. The strongest photoacoustic signal is obtained upon excitation at 905 nm, where there is no noticeable absorbance of either glucose or water, and the near-infrared absorbing dye Acid Black 2 (Acrose Organics, NJ, Pittsburgh, PA) , Fischer Scientific supplied water-soluble nigrosine) showed a large absorbance value at 905 nm.

The PA signal intensity in FIG. 5 increased 6 times in the presence of the external light absorber nigrosine dye as compared with the case of pure water shown in FIG. The ultrasonic pulse amplitude at 905 nm as a function of glucose concentration showed the best response compared to zero response with pure water in FIG. The PA signal of glucose concentration fits a linear equation, PA (mv) at 905 nm = 79.246 [G g / dL] +44.144. The gradient was 79 mV per gram of glucose (r ² = 0.997). The pulse amplitude of water at a wavelength of 1459 nm is PA (mv) at 1459 nm = 16.3 [G g / dL] +44.144, R ² = 0.965, and the slope is close to the slope for water in FIG. It was. The pulse amplitudes at 1550 nm and 1649 nm of the glucose wavelength were PA (mv) = 5.6 [G g / dL] +5.4, r2 = 0.92, which was the same as in the case of the aqueous solution containing no dye. . The PA signal with glucose or water was unaffected by the presence of a highly absorbing nigrosine dye having its maximum absorbance at 905 nm. The strong photoacoustic signal at the nigrosine dye absorption wavelength is due to the added external absorber, in this case nigrosine, absorbing light to produce a photoacoustic pulse, which is the internal thermoelastic property of the solution, i.e. It again suggests that it was modulated by the effect of glucose concentration on the values of β, v and C _p in equations (1) and (2). Thus, prior art photoacoustic spectroscopy studies have neglected the role played by the influence of glucose or other solutes on the internal thermoelastic properties of the medium. Prior art photoacoustic spectroscopy does not take into account the effect of glucose concentration on the thermoelastic properties of the tissue, and thus on the intensity of the detected ultrasonic pulse amplitude, and converts the photoacoustic signal to solute molecules such as glucose molecules. It was supposed to be due to light absorption by.

A third example is shown in FIG. The photoacoustic signal of a suspension of fixed red blood cells made from dog blood and suspended in 0.9% sodium chloride solution containing a variable concentration of glucose is plotted against the glucose concentration. . The photoacoustic signal plotted as a function of glucose concentration at 905 nm showed a slope that fits a linear least square of 17.3 mV per gram of glucose (r ² = 0.995). The photoacoustic signal at a wavelength of 1459 nm for water shows a slope of 18.5 mV per gram of glucose (r ² = 0.994), and the photoacoustic signal at the glucose wavelengths of 1550 nm and 1649 nm is about 5 per gram of glucose. It showed the same gradient of .9 mV (r ² = 0.904). The strong photoacoustic signal at the hemoglobin absorption wavelength of 905 nm again shows that the fixed red blood cells acted as external absorbers when added to the glucose aqueous system. Fixed erythrocytes absorb light and generate a photoacoustic signal, which influences the glucose concentration on the internal thermoelastic properties of the solution, ie, the values of β, v, C _p , D and Λ in equation (3). Modulated by.

The same experiment was repeated using a 1:50 suspension of canine fixed erythrocytes to obtain the PA signal shown in FIG. 6B. The linear equation is
PA (mv) at 905 nm = 16.3 [Gg / dL] +14.747, R ² = 0.983
PA (mv) at 1459 nm = 19.641 [Gg / dL] +7.7765, R ² = 0.995
PA (mv) at 1550 nm = 5.616 [Gg / dL] −3.07, R ² = 0.977
PA (mv) at 1649 nm = 7.283 [Gg / dL] + 4.205, R ² = 0.965
The gradient intensities at 1459 nm, 1550 nm and 1649 nm were similar to the 1: 100 dilution of canine fixed erythrocytes of FIG. 6A, the nigrosine dye of FIG. 5 and the pure water shown in FIG. It was. The glucose PA signal excited at 905 nm and containing a 1:50 suspension of canine fixed red blood cells had a lower slope than that of the 1: 100 dilution of the dog fixed red blood cells of FIG. 6A. Light scattering by the higher concentration suspension may have affected the PA signal at this wavelength.

  Thus, prior art photoacoustic spectroscopy for glucose measurement has neglected the role played by the influence of glucose or other solutes on the internal thermoelastic properties of the medium (tissue). Conventional methods do not separate the influence of glucose as an energy absorber that generates photoacoustic signals that are detected later from the role of glucose as a modifier of the internal thermoelastic properties of the medium or tissue. The prior art does not attempt to correct the contribution of glucose or other analytes of interest to the thermoelastic properties of the medium and thus to the modification of the measured signal. The prior art does not discuss how to correct the effect of bulk tissue properties on the measured signal.

  The prior art describes the use of external or added absorbers in the measurement of the effect of solutes on the internal thermoelastic properties of the medium as shown with nigrosine dye addition or with dog fixed erythrocytes containing hemoglobin. Does not discuss use.

  The prior art also does not discuss the role of medium components in changing the amplitude of ultrasonic pulses generated as a result of adding a light absorber outside the medium. The change in the amplitude of the pulse produced by another absorber (added to the system) is shown in the case of a linear relationship between the photoacoustic signals from the nigrosine dye excited as a function of glucose concentration by the 905 nm laser pulse. ing. Another example is an increase in the photoacoustic signal of water with increasing glucose concentration. A third example of the effect of the solute on the thermoelastic properties of the medium is a linear relationship between the photoacoustic signal of fixed red blood cells and the concentration of added glucose.

  In the in vivo experiments discussed above, an external absorber was added to the medium. In the case of in vivo photoacoustic experiments on the subject site, the external absorber is a dye applied to the skin, a film with a known optical density (OD) that is brought into contact with the skin, or excited as a detector. It may be in the form of a dye incorporated into a polymer coating of PZNT crystals used as a window, or other similar light absorbing material. In this case, a PZNT transducer is used in the reflection mode, where the backpropagated ultrasonic pulse is detected.

The external absorber of the embodiment of the present invention has a known high optical density at the target wavelength. This optical density must be large enough to affect the signal without losing its ability to excite the photoacoustic signal of the light pulse. For example, between 0.5 and 1.0 at all wavelengths used for excitation. At an optical density of 1.0, the added material absorbs more than 90% of the incident light intensity. The external absorber has an optical density higher than 2.0 (ie, absorbs over 99% of the light intensity). When the laser pulse acts on an external light absorber coupled to the tissue at the subject site, the ultrasonic pulse propagates to the subject site. A comparison of the contribution of the natural absorption compound in the tissue and the external absorber to the generated photoacoustic signal is shown in Table 1.

  The photoacoustic signal can be detected in two different arrangements. The first arrangement is an in-line or transmission arrangement in which the excitation light beam and detector are on opposite sides of the subject site. The beam and detector may be in-line or off-line to avoid the possibility of direct light acting on the detector. Another arrangement is a reflective arrangement where both the excitation light beam and the detector are on the same surface of the subject site. This arrangement is used for photoacoustic imaging. The use of PZNT detectors for imaging and analytical applications is disclosed in WO 2004/042382.

  Externally added absorbers are coated with neutral density filters (such as Wratten gelatin filters containing silver particles), exposed and developed photographic or X-ray films, high extinction coefficient near infrared absorbing dyes. Films, carbon black particle coated films, dyes, colored polymer particles and the like. The external light absorber may be contained in the polymer coating material of the single crystal piezoelectric transducer (PZNT). In that case, the PZNT transducer is used in a reflective mode to detect backpropagated ultrasonic pulses.

  The external additional absorber efficiently absorbs most of the energy of the laser pulse and propagates into the subject site to reach the detector, where the internal characteristics of the tissue, the size of the subject site, and the surface Convert to ultrasonic energy that gives a signal about features. The internal characteristics of the tissue, the dimensions of the specimen site, and the surface characteristics, as well as the extraneous structure embedded in the specimen site, are the characteristics of the ultrasound pulse generated under efficient conversion conditions from light pulses to ultrasound pulses. Affects amplitude. It is preferred that the total light energy is absorbed by the outer absorption layer and therefore the propagation signal is constant for the same subject site measured under the same experimental conditions.

(PA) _NA is a photoacoustic signal due to light absorption by naturally occurring absorbing molecules in the subject site, such as hemoglobin, water or glucose. (PA) _NA can be expressed as:

(PA) _NA = RTDΛS. [(Βv ² ) / C _p ] _bulk (μ _a ) _NA E ₀ (4)
However, (μ _a ) _NA is a term representing the light absorption of the target analyte, such as hemoglobin, water or glucose, and (μ _a ) _NA in formula (4) is a change in the concentration of this analyte. Represents the effect of the on the light absorption process and photoacoustic signal generation. The second term of equation (4), expressed as ((βv ² ) / C _p ) _bulk , represents the internal thermoelastic properties of the medium, and the RTDΛS product represents the acoustic propagation within the tissue.

When an external absorber is added that has an OD or extinction coefficient higher than the analyte that needs to be measured, the resulting signal is expressed by Equation 5. (PA) _EA is a photoacoustic signal generated in the presence of an added external absorber. It is expressed as follows:

(PA) _EA = RTDΛS [(βv ² ) / C _p ] _bulk (μ _a ) _EA . E ₀ (5)
However, (μ _a ) _EA is a term representing the light absorption of the added external light absorber. It has a deliberately high absorbance and is therefore distinct from the effect of this analyte concentration on the absorption process in the tissue and the generation of photoacoustic signals. Another term, expressed as RTDΛS and [(βv ² ) / C _p ] _{bulk in} equations (4) and (5), represents the acoustic propagation in the tissue and the internal thermoelastic properties of the tissue, Same as the case.

  Prior art photoacoustic signal formulas for irradiation energy, absorption, compression wave generation and ultrasonic pulse detection are coupled with full efficiency to the tissue and the generated compression wave to the tissue interface. As a result, the coupling to the detector is assumed. In a more descriptive equation, a multiple factor γ should be introduced into equations (4) and (5) to take into account their coupling efficiency, which results in the following equation:

(PA) _NA = _γNA RTDΛS [(βv ² ) / C _p ) _bulk ] (μ _a ) _NA . E ₀ (6)
and,
(PA) _EA = _γEA RTDΛS [(βv ² ) / C _p ] _bulk ] (μ _a ) _EA . E ₀ (7)
Where _γNA and _γEA are the coupling efficiency terms of the two photoacoustic measurements.

  An important aspect of the method of embodiments of the present invention is the external absorber. The purpose is not to attenuate the light intensity applied to the skin, but to absorb the light intensity based on the following characteristics and convert it into an acoustic signal.

1) The external absorber has a much higher extinction coefficient than the analyte to be measured.
2) The external absorber is extremely thin, and the dye layer on the skin is several micrometers thick. Another form of external light absorber is a thin film having a light absorber on top. The thickness of the film and the light absorber is about 100 μm.
3) The external absorber is efficiently bound to the tissue, in this case the skin, and the binding is affected by the use of ultrasound and heat transfer gels or adhesives.
4) The external absorber itself has no internal characteristics. Instead, it absorbs light and converts it to heat, which leads to the generation of pressure waves in the tissue to which it is attached.
5) Transmission of mechanical stress to the tissue causes the propagation of the resulting ultrasonic pulse to be controlled by the internal characteristics of the tissue.

Thus, the term [(βv ² ) / C _p ) _bulk ] and the terms R, D, and Λ in equation (7) relate to the same bulk texture properties in equation (6). Excitation of the photoacoustic signal and transmission of mechanical stress to the tissue in an external absorber coupled to the tissue is performed with a low energy laser pulse that does not pierce the absorber layer or skin by thermal melting. Is done. The use of strong absorbers and laser pulses on the skin to puncture the skin was described by Epstein et al. In US Pat. No. 5,858,211. Thus, an object of embodiments of the present invention is to excite photoacoustic signals in tissue while preventing damage to the skin, removal of the stratum corneum, or opening of holes in the skin.

Next, using the two measurement signals generated at each wavelength (PA) _NA and (PA) _EA , the PA in the natural state is divided by the intensity of the photoacoustic signal when an external absorber is added, and the ratio (PA ) _NA / (PA) _EA was calculated. The resulting ratio is the ratio of the normalized signal generated from the absorbing species in the tissue (hemoglobin, water and glucose) to the signal generated at a higher absorbance and converted to ultrasound energy. The ratio of the photoacoustic (natural state) to the photoacoustic (external light absorber) at each wavelength is further used in the calculation shown below.

Dividing equation (6) by equation (7) gives
_{[(PA) NA / (PA} ) EA] = (γNA / γEA). [(Μ _a ) _NA / (μ _a ) _EA ] (8)
The coupling efficiency of equation (8) for a given instrument and experimental condition can be expressed as:
Γ = ( _γNA / _γEA ) (9)
_Substituting ( _γNA / _γEA ) and extinction coefficient as an external absorber,
(Μ _a ) _EA = 2.303 [(OD / l) _EA (10)
When a dye added to the sample or applied to the sample surface is used, the absorption coefficient of the external light absorber can be expressed by Equation 10a.
(Μ _a ) _EA = 2.303ε _NA C _NA (10a)
The optical parameter OD in the equation (10) is the optical density of the added external absorber at the same wavelength λ, and l is the thickness of the natural absorber. Substituting these expressions in equation (9) yields equation (11).
_{[(PA) NA / (PA} ) EA] λ = Γ [(μ a) NA /2.303[(OD/l) EA] λ (11)
Equation (11) can be used to determine the extinction coefficient of the natural light absorber (μ _a ) _NA , where the natural light absorber is selected from the group of molecular metabolites including water, glucose, hemoglobin, and other equivalents. One. The natural light absorber may be a chemotherapeutic or phototherapeutic or other field drug that is important for tracking its concentration in the tissue.

The extinction coefficient of the natural light absorber is expressed as follows.
(Μ _a ) _NA = 2.303ε _NA C _NA (12)
ε _NA is the molar extinction coefficient (or molar absorption coefficient) of the naturally occurring analyte at wavelength λ, and C _NA is the molar concentration of the natural absorber, Guided to the formula.
[(PA) _NA / (PA) _EA ] _λ = Γ. [2.33ε _NA C _NA /2.303 [(OD / l) _EA ] _λ (13)
The spontaneous absorber, i.e., the molar concentration of the control analyte in the tissue, will then be expressed as:
C _NA = Γ ⁻¹ . [(PA) _NA / (PA) _EA ] _λ . ^{_{[(OD / l) EA ~}} / ε NA] λ (14)
Incidentally, _{"A ^~"} is a notation indicating that is disposed "~" on top of "A". This equation represents the absolute molar concentration of a naturally occurring absorber (such as water, hemoglobin, glucose or the like) in the tissue. The spontaneous analyte molar concentration in the tissue is expressed in relation to:
a. Photoacoustic signal from a naturally occurring absorber
b. Photoacoustic signal in the presence of an added external absorber
c. Optical density of added external absorber
d. The thickness of the added absorption layer
e. The extinction coefficient of spontaneous absorbers at the same wavelength The concentration is changed from moles per liter to conventional clinical concentration units such as mg / dL of glucose, g / dL of hemoglobin, or% of water in the tissue. May be. The method of the embodiment of the present invention is shown in the flowchart of FIG.

If the coupling efficiency of the first measurement _γNA when exciting the naturally occurring _absorber is the same as the second measurement _γEA when exciting the external absorber, the coupling efficiency term Γ of Equation 14 is Γ = (Γ _NA / γ _EA ) = 1, and Equation (14) is simplified below.

C _NA = [(PA) _NA / (PA) _EA ] _λ . ^{_{[(OD / l) EA ~}} / ε NA] λ (15)
The coupling efficiency term Γ incorporates the pressure of the measurement device on the subject site, the collection efficiency of the ultrasonic detector, the irradiation efficiency of the laser light source and the optical element. Pressure control, relocation variations, and other experimental parameters are extremely stringent to achieve the conditions that lead to equation (15). Alternatively, the binding efficiency parameter Γ is experimentally measured to determine the concentration of the naturally occurring analyte _CNA .

An embodiment of the present invention is a method for measuring glucose in human tissue, and includes the following steps.
a. Ultrasound is generated in human tissue by irradiating a subject site with a near-infrared short-time laser pulse.
b. A first signal as an ultrasonic pulse obtained at each wavelength is detected.
c. The same laser wavelength and energy are used, and a light-absorbing material is inserted between the laser source outside the tissue and the tissue, and the light-absorbing material is inserted into the tissue, and an ultrasonic pulse is generated in the light-absorbing material to penetrate the tissue Then, an ultrasonic pulse is generated in the same tissue so as to propagate to the ultrasonic detector.
d. The second signal obtained in the presence of the external light-absorbing substance is detected as an ultrasonic pulse generated at each wavelength.
e. At each irradiation wavelength, the ratio of the intensity of the pulse generated in step (b) to the intensity of the pulse generated in step (d) is calculated.
f. The extinction coefficient of glucose in the tissue is calculated from the ratio of the intensity of the signal obtained in step (e) and the predetermined optical characteristics of the external light-absorbing substance.
g. The concentration of glucose is calculated from the extinction coefficient calculated in step (f), and the molar extinction coefficient of the analyte at the photoacoustic excitation wavelength is calculated.

  In the case of noninvasive measurement of glucose by the photoacoustic analysis method of the embodiment of the present invention, a near-infrared wavelength with higher glucose absorbance, such as 1660 nm, is used as the excitation wavelength. Substituting a value into equation (14) yields:

C _glucose = Γ ⁻¹ . _{[(PA) NA / (PA} ) EA] @ 1660nm [(OD / l) EA ~ / ε glucose] @ 1660nm (16)
A series of photoacoustic measurements were performed at different blood glucose concentrations, such as a food tolerance test or an oral glucose tolerance test. C _glucose vs. [(PA) _NA / (PA) _EA ] _{@ 1660nm} was plotted and the slope of the plot was calculated to be the same as Γ- ¹ .

  Another aspect of the method of embodiments of the present invention is a method for measuring glucose in the human body, but the external absorber also has a higher extinction coefficient than glucose at at least one wavelength, and is naturally occurring in the human body. Molecule. Water in the human body can also be used as an external light absorber. In this case, at least two laser wavelengths are used, one of which is in the near-infrared absorption band of water, and the second is the overtone of glucose in the near-infrared region or the absorption band of the combined sound. Is the wavelength.

Another aspect of the method of embodiments of the present invention is a photoacoustic method that uses water as an external absorber and measures glucose in human tissue using at least two laser excitation wavelengths. This is one method for measuring C _glucose and Γ by performing multiple wavelength measurements with the same measurement. In this experiment, the PA signal of the natural absorber (glucose) and the PA signal of the external absorber (water) are measured (at least) at two wavelengths. One wavelength is selected for the glucose absorption wavelength, for example 1660 nm. The second wavelength is selected as the water absorption wavelength, such as 1250 nm, 1400 nm, 1450 nm, 1459 nm. Then, equation (14) is written as follows.

C _glucose = Γ ⁻¹ . _{[(PA) NA / (PA} ) EA] @ 1660nm [(OD / l) EA / ε glucose] @ 1660nm (16)
C _water = Γ ⁻¹ . [(PA) _NA / (PA) _EA ] _{@ 1450 nm} [(OD / l) _EA / ε _water ] _{@ 1450 nm} (17)
Due to the strict electrolyte balance maintained in the normal human body, the water content of the tissue is considered to be slowly changing and constant, with the exceptions of dehydration, edema, renal failure and so on. It can be assumed that the moisture is constant and about 70% of the tissue weight. Therefore, the concentration of water in the tissue is 0.70 × 55 = 38.5 mol per liter. Substituting into equation (17) results in two equations (18) and (19).

38.5 = Γ ⁻¹ . [(PA) _NA / (PA) _EA ] _{@ 1450 nm} [(OD / l) _EA / ε _water ] _{@ 1450 nm} (18)
Γ ⁻¹ = 0.2579 [(PA) _NA / (PA) _EA ] _{@ 1450 nm} [(OD / l) _EA / ε _water ] _{@ 1450 nm} (19)
And as a result,
_{C glucose = {0.2579 [(PA} ) NA / (PA) EA] @ 1450nm [(OD / l) EA / ε water] @ 1450nm} -1 x {[(PA) NA / (PA) EA] @ _{1660 nm} [(OD / l) _{EA /} ε _glucose ] _{@ 1660 nm} } (20)
C _glucose = 38.5 {[(PA) _EA / (PA) _NA ] _{@ 1450nm} [(PA) _NA / (PA) _EA ] _{@ 1660nm} } x [ε _{water @ 1450nm} / _{εglucolose @ 1660nm} ] x [(OD / L) _{EA @ 1660nm} / (OD / l) _{EA @ 1450nm} (21)
C _glucose = 38.5 [(PA) _{NA @ 1660nm} / (PA) _{NA @ 1450nm} ] [(PA) _{EA @ 1450nm} / (PA) _{EA @ 1660nm} ] x [ _{εwater @ 1450nm / εglucocolse @ 1660nm} ] x [ (OD / l) _{EA @ 1660nm /} (OD / l) _{EA @ 1450nm} ] (22)
Intensity of photoacoustic signal amplitude for natural light absorber (glucose) [(PA) _{NA @ 1660} / (PA) _{NA @ 1450} ], value of molar extinction coefficient of glucose at one wavelength, absorption of water at another wavelength The glucose concentration in the tissue is calculated from the value of the coefficient and the optical density of the external absorber (water) at two wavelengths. Expression (22) is applied to a subject without dehydration and without edema or renal failure.

  Another aspect of embodiments of the present invention is a method for measuring glucose concentration in tissue using six photoacoustic signals at three wavelengths. Three of these measurements are performed to obtain signals at the water and glucose excitation wavelengths in the standard tissue. The other three measurements are performed in the presence of an external light absorbing material in contact with the tissue as described above. Using multiple wavelengths, including the water excitation wavelength, and an external absorber allows for NIR measurement of glucose without any estimation of the body's steady moisture. In this case, it may be possible to measure glucose even in the presence of dehydration or edema.

  Thus, in a non-invasive measurement of glucose concentration in a tissue, the measurement must be performed at (at least) three wavelengths without making some kind of estimation of tissue moisture. One of these wavelengths is the wavelength at which water has significant NIR absorbance. One wavelength selection scheme that can be envisioned is to select wavelengths of 1550 nm and 1660 nm for glucose and 1450 nm for water. Then, equation (14) is written as equations (23), (24) and (25).

C _glucose = Γ ⁻¹ . [(PA) _NA / (PA) _EA ] _{@ 1550nm} [(OD / l) _{EA / εglucose} ] _{@ 1550nm} (23)
C _glucose = Γ ⁻¹ . _{[(PA) NA / (PA} ) EA] @ 1660nm [(OD / l) EA / ε glucose] @ 1660nm (24)
C _glucose = Γ ⁻¹ . [(PA) _NA / (PA) _EA ] _{@ 1450 nm} [(OD / l) _{EA /} ε _water ] _{@ 1450 nm} (25)
Solving these equations simultaneously yields the glucose glucose concentration C _glucose in the tissue. In this case, one external absorber is used and the ultrasonic pulse amplitude is measured at three near infrared wavelengths. Two of these wavelengths are glucose absorption wavelengths and the third is in the near infrared water absorption band. The values of ε _water and ε _glucose are measured from independent near-infrared measurements using, for example, a Fourier transform NIR instrument in an aqueous medium at the same temperature (37 ° C.) as the tissue temperature.

  Alternatively, the ultrasonic pulse amplitude is measured at two water absorption wavelengths and one glucose absorption wavelength, resulting in a variation of equation (11) of the form

C _water = Γ ⁻¹ . _{[(PA) NA / (PA} ) EA] @ λ1water [(OD / l) EA / ε water] @ λ1water (26)
C _water = Γ ⁻¹ . _{[(PA) NA / (PA} ) EA] @ λ2water [(OD / l) EA / ε water] @ λ2water (27)
C _glucose = Γ ⁻¹ . _{[(PA) NA / (PA} ) EA] @ λglucose [(OD / l) EA / ε Glucose] @ λglucose (28)
These three equations are solved in terms of the molar concentration of _glucose C _glucose and the molar concentration of _water C _water . The molar concentration of water can be used to assess the hydration state of the tissue. The experimental measurement is a two-stage experiment with one OD value of the external absorber and three laser irradiation wavelengths.

  Accordingly, another embodiment of the present invention is a photoacoustic analysis method for measuring tissue hydration and glucose concentration in the tissue. The experimental measurement is a two-stage experiment with one optical density (OD) value of the external absorber and three laser irradiation wavelengths.

Another embodiment of the invention is a photoacoustic analysis method for measurement of glucose in tissue. This method for measurement of C _glucose in tissue involves the use of two optical density values of the external absorber and two illumination wavelengths. In this method, three photoacoustic measurements are performed.

Step (1) Ultrasonic pulse amplitude measurement in the natural state with no external absorber in the optical path results in (PA) _NA at glucose and water wavelengths.

Step (2) An external absorber layer whose optical density per cm is (OD / l) _EA1 is inserted between the light source and the subject site, and the second ultrasonic pulse amplitude measurement is performed at two wavelengths, (PA) _EA1 at the wavelength of glucose and water was obtained.

Step (3) Optical density per cm is different (OD / l) The second layer external absorber of _EA1 is inserted between the light source and the tissue, and the third ultrasonic pulse amplitude measurement is performed at two wavelengths And obtained (PA) _EA2 at the wavelength of glucose and water. The following three formulas (29), (30), and (31) were generated and solved to obtain the molar concentration of _glucose , C _glucose .

^{_{C glucose = Γ -1 [(PA}} ) NA / (PA) EA1] @ λglucose [(OD / l) EA1 / ε glucose] @ λglucose (29)
^{_{C glucose = Γ -1 [(PA}} ) NA / (PA) EA2] @ λglucose [(OD / l) EA2 / ε glucose] @ λglucose (30)
C _water = Γ ⁻¹ [(PA) _NA / (PA) _EA1 ] @ _λwater [(OD / l) _{EA1 /} ε _water ] _{@ λwater} (31)
The formula was solved to determine the molar concentration of glucose in the tissue. Applying the same procedure, it is possible to measure the molar concentration of hemoglobin, lipid or water in the tissue by inserting the appropriate use of the irradiation wavelength and the appropriate molar extinction coefficient and optical density for the external absorber. is there. Water and hemoglobin measurements help assess a patient's hydration and anemia status along with the patient's blood glucose status. Measurement of these three parameters is important, for example, for the treatment of elderly patients who tend to easily become anemic or dehydrated while suffering from diabetes. Thus, the methods of embodiments of the present invention provide a more advanced follow-up of the health status of these patients who are at risk of developing dehydration or anemia in addition to their own diabetes.

In all embodiments of the invention,
1. At least one of the laser wavelengths is a wavelength specific to the analyte.
2. At least one of the wavelengths is a wavelength characteristic of water.
3. The laser pulse duration should be between 10 ^{−9 and} 10 ⁻⁶ seconds.
4). The detector is a piezoelectric crystal (PZT, lead, zirconium, tantalum crystal), hydrophone or polyvinylidene fluoride (PVDF) film.
5. The detector is a piezoelectric crystal (PZNT, lead, zirconium, nubium, tantalum crystal).
6). The detector is coated with a polymer layer to match the acoustic impedance characteristics of the tissue.
7). The external light absorber is a film coated with a light absorption coefficient light-absorbing substance such as dispersed carbon black particles.
8). The external light absorber is a film coated with a light absorption coefficient light-absorbing substance such as a dispersed high-concentration dispersive light-absorbing dye.
9. The external light absorber is a film coated with a light absorption coefficient light-absorbing substance such as a dispersive light-absorbing dye.
10. The external light absorber is a film coated with a light absorption material having a high extinction coefficient such as a dispersion-colored polymer material.

The following embodiments of the present invention will be described in detail.
First Embodiment Photoacoustic Response of Aqueous Glucose Solution in the Presence of External Absorber Acid Black 2 The method of the embodiment of the present invention was tested using the instrument set described in FIG. As an in vitro experiment, the sample was placed in a quartz cell with one side in contact with a PZNT ultrasonic detector coated with a polymer layer. A 1 μm thick gold film was placed to prevent the laser beam from acting directly on the detector in the transmissive arrangement. A glucose solution (0-20 g / dL) was prepared. The glucose solution was placed through the screw cap into a centrifuge vial with two connected Teflon® tubes (Teflon® Tube). The vial was placed in a constant temperature water path and was connected to a 1CM quartz flow cell and a peristaltic pump. The sample was circulated during the measurement. FIG. 3 shows the photoacoustic signal as a function of the concentration of glucose irradiated at 905 nm, 1459 nm, 1550 nm and 1649 nm and added to the medium. Three lines were obtained from plots of the signal for glucose at 1459 nm, 1550 nm and 1649 nm. 1459 nm is the absorption wavelength of water, and 1550 nm and 1649 nm are the absorption wavelengths of glucose. When the aqueous glucose solution was excited below 905 nm, there was no photoacoustic signal detectable.

The effect of the added external absorber on the photoacoustic signal obtained from the change in glucose concentration was examined. Solutions with different glucose concentrations were prepared with 0.9% sodium chloride solution containing a fixed amount of near infrared absorbing dye. The near infrared absorbing dye was Acid Black 2 (water-soluble nigrosine from Acrose Organics, NJ, supplied by Fisher Scientific, Pittsburgh, PA). All solutions had an extinction coefficient μ _a = 2.0 CM ^{−1 at} 905 nm (2 units optical density per cm at 905 nm). The sample was circulated in a 1 cm quartz flow cell as in the case of a pure aqueous solution.

PA signals were recorded at 905 nm, 1459 nm, 1550 nm and 1649 nm. As shown in FIG. 5, four linear response plots were obtained as a function of glucose concentration. The strongest photoacoustic signal linearly related to glucose concentration is obtained from excitation at 905 nm, where neither glucose nor water has appreciable absorbance. It nigrosine dyes of the external light-absorbing body, a wavelength having a mu _a value of 2.0 cm ^-1. It is clear that the dye absorbed light and that the PA signal amplitude due to nigrosine dye excitation was modulated by the linear relationship of glucose to the internal thermoelastic properties. As shown in FIG. 5, the intensity of response to changes in glucose concentration was highest at the excitation wavelength of the external absorber added to the glucose solution and lowest at the glucose absorption wavelength. Thus, the use of an external light absorber enhances the sensitivity of the photoacoustic signal to changes in glucose concentration in the aqueous medium.

Second Embodiment Photoacoustic Response of Aqueous Glucose Solution in the Presence of Dog Fixed Red Blood Cells as External Absorber Additional External Absorption that Mimics Human Blood Spectrum for Photoacoustic Signal Obtained from Glucose Concentration Change in Aqueous Medium I examined the effects of the body.

  Solutions with different glucose concentrations were prepared in 0.9% sodium chloride solution containing a 1: 100 dilution of canine fixed erythrocytes. Here, fresh dog red blood cells were tested, but the data were not reproducible due to the osmotic effect of glucose on red blood cells. Microscopic examination showed red blood cell contraction and malformation at high glucose concentrations. Dog erythrocytes were fixed using a glutaraldehyde solution, spun and washed. Fixed erythrocytes are polymer sac containing hemoglobin, and their characteristics do not change over time. The hemoglobin in the added red blood cell serves as an external absorber added to the system.

  PA signals were recorded at 905 nm, 1459 nm, 1550 nm and 1649 nm. As shown in FIG. 6A, four linear response plots were obtained as a function of the glucose concentration shown for a 1: 100 suspension of canine fixed red blood cells in glucose solution. The strongest photoacoustic signal linearly related to glucose concentration is obtained from excitation at 905 nm, where neither glucose nor water has appreciable absorbance. It is the wavelength at which hemoglobin of fixed erythrocytes, which is an external absorber, has absorbed light. It is clear that hemoglobin absorbs light at 905 nm and that the PA signal amplitude due to hemoglobin excitation was modulated by the linear relationship of glucose to internal thermoelastic properties. As shown in FIG. 6A, the intensity of response to changes in glucose concentration was highest at the excitation wavelength of the external absorber added to the glucose solution, and lowest at the absorption wavelength of glucose. Thus, the use of an external light absorber that mimics human blood in the tissue enhances the sensitivity of the photoacoustic signal to changes in glucose concentration in the aqueous medium.

  The experiment was repeated using a 1:50 suspension of canine fixed erythrocytes as the external absorber. The behavior of the photoacoustic signal as a function of glucose concentration was similar to the 1: 100 suspension. The 905 nm wavelength was higher for pure water, ie, zero, but less than for the 1: 100 suspension. Scattering by higher concentrations of fixed red blood cells affected the signal. The effect of scattering on the PA signal has been reported in the art. The data is shown in FIG. 6B.

(Third Embodiment) Photoacoustic response of the subject site before and after adding the black polymer film as the external absorber In order to investigate the effect of the added external absorber on the photoacoustic signal generated in the subject site, The photoacoustic analysis system shown in FIG. 2A was used. It is a four-pulse solid laser of 905 nm, 1459 nm, 1550 nm and 1649 nm. The light from each laser was delivered to the lens housing by optical fiber. The lens focused the laser pulse on the human finger.

  The subject placed his hand on the optical element table and placed his finger between the lens housing (26) and the PZNT detector (32) and sat on the laboratory stool. The detector is a PZNT single crystal detector manufactured by Toshiba Medical Systems Co., Ltd. (Tokyo, Japan), coated with an impedance compatible polymer film to minimize ultrasonic coupling loss between the detector and the skin. It is a thing. An ultrasonic coupling gel was applied to the side of the finger in contact with the detector. The signal from the PZNT detector was amplified and routed to a Tektronix digital storage scope. The laser trigger was controlled by a personal computer using LabView software. The trigger pulse from the laser trigger module triggered data acquisition by the digital storage scope. The time evolution of the pressure transient signal, ie the detected ultrasound signal, was recorded. Two parameters were extracted from the data at each photoacoustic excitation wavelength. First, the delay time between the laser trigger pulse and the first peak of the pressure transient signal. This time varies from 5 μs to 15 μs depending on the thickness of the sample and its opacity. The second parameter is the amplitude from the highest to the lowest recording pulse.

  7A, 7B, 7C and 7D show PA signals generated from human fingers excited at 905 nm, 1459 nm, 1550 nm and 1649 nm, respectively. The solid lines of the four graphs up to FIGS. 7A, 7B, 7C, and 7D represent signals generated by finger excitation in the natural state. The amplitude of the pulse varied from 350 mV to 800 mV depending on the excitation wavelength.

  Next, an external absorbent film was applied between the lens and the finger. The external absorber is an 80 μm thick polyethylene terephthalate (PET) film in which minute carbon black is diffused. The carbon content in the adhesive was 2% acetylene black. The absorption spectrum of the acetylene black coated film was performed on a near infrared FTIR instrument (Bruker, Equinox 55 / S spectrometer). The optical densities of the external absorbers at the four excitation wavelengths were 4.12 at 905 nm, 3.2 at 1459 nm, 3.12 at 1550 nm, and 3.04 at 1649 nm.

  A small piece of PET film (external light absorber) was cut off, the polymer backing was peeled, and then the film whose adhesive side touched the skin was pressed against the skin to achieve efficient bonding to the skin. An ultrasonic coupling gel was applied to the side of the finger that contacted the detector. The finger was placed between the lens holder and the detector so that the lens holder pressed the film and the detector pressed the skin on the opposite side of the finger. The stress between the lens housing and the detector applied to the skin was 200 g.

  The solid lines in the four graphs up to FIGS. 7A, 7B, 7C, and 7D represent signals generated by excitation of the finger and an additional PET film having acetylene black as an external absorber. The photoacoustic signal was excited at 905 nm, 1459 nm, 1550 nm and 1649 nm. The amplitude of the pulse varied from 560 mV to 4700 mV depending on the excitation wavelength of the photoacoustic signal. An important observation is the increase in photoacoustic signal amplitude at all wavelengths. Regarding the subject's finger in this embodiment, the photoacoustic signal saw the following enhancement by the addition of an external absorber. The signal at 905 nm excitation increased from 600 mV to 4700 mV, the signal at 1459 nm excitation increased from 800 mV to 1700 mV, the signal at 1550 nm excitation increased from 350 mV to 560 mV, and the signal at 1649 nm excitation increased from 350 mV to 660 mV. The highest increase in the amplitude of the PA signal was obtained at 905 nm where the external absorber has the highest absorbance. The results of this experiment suggest that it is possible to bind the light-absorbing film to the skin of a human finger and generate a photoacoustic signal. The enhanced signal is absorbed by light with a high optical density external absorber that is coupled to the skin of the human finger, and ultrasound is generated in the finger tissue and transmitted through the finger. This is due to being detected by a piezoelectric detector coupled to the opposite side of the finger by a gel.

  The behavior of the PA signal in the presence of an external absorber was the same as in an in vitro experiment with an aqueous glucose solution. Those skilled in the art can measure glucose concentration or change glucose concentration in the human body using signal intensity, extinction coefficient of absorber and glucose at various wavelengths, and the equations described in the detailed description of embodiments of the present invention. Can be measured. Those skilled in the art will dissociate the signal obtained from natural absorption measurements and measurements in the presence of external absorbers from the essence of the present invention using data reduction routines other than linear least squares or advanced data processing methods. Can be processed without any problems.

  Note that the present invention is not limited to the above-described embodiment as it is, and can be embodied by modifying the constituent elements without departing from the scope of the invention in the implementation stage. In addition, various inventions can be formed by appropriately combining a plurality of components disclosed in the embodiment. For example, some components may be deleted from all the components shown in the embodiment. Furthermore, constituent elements over different embodiments may be appropriately combined.

2 shows a flow chart illustrating a method of an embodiment of the present invention. The structural example of the photoacoustic analysis system used with the method of embodiment of this invention is shown. The other example of a structure of the photoacoustic analysis system used with the method of embodiment of this invention is shown. In this embodiment, the photoacoustic signal produced | generated from the glucose solution in a 1 cm quartz cell is shown. In the present embodiment, infrared absorbance of an aqueous glucose solution at 1459 nm, 1550 nm, and 1649 nm is shown. In this embodiment, PA signals of various concentrations of glucose are shown in the presence of the well-known attached absorber (acid optical 2 dye at 905 nm, 2.0 optical density units). In this embodiment, 2% dog fixed blood cells (2.0 optical density units at 905 nm) are used as well-known attached absorbers, and PA signals of various concentrations of glucose in the presence of 1: 100 dilution of saline. Show. In this embodiment, the well-known attached light absorber, 2% canine fixed blood cells (2.0 optical density units at 905 nm), PA signals of various concentrations of glucose in the presence of 1:50 dilution of saline. Show. A photoacoustic signal generated from a human finger excited at 905 nm is shown (solid line). A broken line is a signal in a polyethylene terephthalate (PET) film having carbon particles as a light absorber, which is an added external light absorber. The OD of the film at 905 nm was 4.2. A photoacoustic signal generated from a human finger excited at 1459 nm is shown (solid line). A broken line is a signal in a PET film having carbon particles as a light absorber, which is an added external light absorber. The OD of the film at 1459 nm was 3.2. A photoacoustic signal generated from a human finger excited at 1550 nm is shown (solid line). A broken line is a signal in a PET film having carbon particles as a light absorber, which is an added external light absorber. The OD of the film at 1550 nm was 3.12. A photoacoustic signal generated from a human finger excited at 1649 nm is shown (solid line). A broken line is a signal in a PET film having carbon particles as a light absorber, which is an added external light absorber. The OD of the film at 1649 nm was 3.04.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 10 ... Apparatus, 11 ... Light source module, 12 ... Power supply, 15, 16, 17, 18 ... Laser source, 20, 21, 22, 23 ... Quartz optical fiber, 25 ... Body part interface module, 28 ... Sample, 27 ... Condensing Lens, 26 ... Lens housing, 32 ... Piezoelectric detector, 34 ... Holder, 35 ... Mechanism, 40 ... Detection electronics / module, 41 ... Disconnection connector, 42 ... Amplifier, 43 ... Digital storage scope, 44 ... Control PC, 46 ... signal line, 48 ... signal line, 50 ... signal line.

Claims (19)

In the photoacoustic analysis method for compensating for the influence of the internal characteristics of the tissue on the signal obtained by measuring the analyte in the target tissue of the subject site,
Generating an ultrasonic pulse in the target tissue by irradiating the subject site with a near-infrared laser pulse from a laser source; and
(B) detecting the ultrasonic pulse using an ultrasonic detector disposed in the vicinity of the subject site and generating a first signal;
A period in which the light-absorbing material is disposed between the laser source and the subject site so that the ultrasonic pulse is generated in the light-absorbing material, propagates through the tissue, and is detected by the ultrasonic detector. And (c) generating an ultrasonic pulse in the target tissue using a laser pulse having the same laser wavelength and energy as in step (b),
Detecting an ultrasonic pulse generated by the light-absorbing material and generating a second signal (d);
Calculating the ratio of the first signal generated in step (b) and the second signal generated in step (d) for each laser wavelength;
A step (f) of calculating an extinction coefficient of the analyte from the ratio calculated in the step (e) and a predetermined optical characteristic of the light-absorbing substance;
A photoacoustic analysis method comprising: (g) calculating a concentration of the analyte from the extinction coefficient calculated in the step (f), and calculating a molar extinction coefficient of the analyte at a photoacoustic excitation wavelength.   The method of claim 1, wherein the analyte is glucose.   The method of claim 1, wherein the analyte is tissue fluid.   The method according to claim 1, wherein the analyte is hemoglobin.   The method of claim 1, wherein the analyte is body fat.   The method of claim 1, wherein the analyte is a chemotherapeutic contrast agent or a diagnostic contrast agent.   The method of claim 1, wherein at least one of the laser wavelengths is a wavelength characteristic of glucose.   The method of claim 1, wherein at least one of the laser wavelengths is a wavelength characteristic of water. The method according to claim 1, wherein the laser pulse has a duration from several 1 × 10 ⁻⁹ seconds to 1 × 10 ⁻⁶ seconds.   The method according to claim 1, wherein the detector is one of a piezoelectric crystal (PZT, lead, zirconium, tantalum crystal), a lithium niobate crystal, a hydrophone, or a polyvinylidene fluoride (PVDF) film.   The method of claim 1, wherein the detector can be a piezoelectric crystal (PZNT, lead, zirconium, nubium, tantalum crystal). The detector is a piezoelectric crystal in the form of a single crystal containing lead titanate, which can be represented by the general formula Pb [(B1, B2) _1-x Ti _x ] O ₃ and has a fraction x = 0.5 to 0.55, B1 represents an element selected from Zn, Mg, Ni, Sc, In and Yb, and B2 represents one element selected from niobium, Nb, tantalum and Ta Item 12. The method according to Item 11.   The method of claim 1, wherein the detector is coated with a polymer layer to match the acoustic impedance characteristics of the tissue.   2. The method according to claim 1, wherein the external additional absorber is a film coated with a light absorbing material having a high extinction coefficient including dispersed carbon black particles.   2. The method according to claim 1, wherein the external absorber is a film coated with a light-absorbing substance having a high extinction coefficient including a high-concentration dispersive light-absorbing dye.   The method according to claim 1, wherein a predetermined pressure is applied to the subject region by a measurement probe. In the photoacoustic analysis method for compensating the influence of the internal characteristics of the tissue on the signal obtained by measuring glucose in the subject tissue,
A step of generating an ultrasonic pulse by irradiating a target site of the subject with a near-infrared laser pulse from a laser source , and detecting a first ultrasonic pulse obtained at each wavelength with an ultrasonic detector (a )When,
A period in which the light-absorbing material is disposed between the laser source and the subject site so that the ultrasonic pulse is generated in the light-absorbing material, propagates through the tissue, and is detected by the ultrasonic detector. And (b) generating a second ultrasonic pulse in the target tissue using a laser pulse having the same laser wavelength and energy as in step (a),
An intensity ratio between the first ultrasonic pulse generated in the step (a) and the second ultrasonic pulse generated in the step (b) is calculated for each irradiation wavelength, and the step Generating a normalized analyte-related photoacoustic signal by compensating for the influence of the bulk tissue property of the subject site on the measurement value of the first ultrasonic pulse generated in (a) (C),
Using the intensity ratio of the ultrasonic pulse, the absorption coefficient value of glucose at two wavelengths, the absorption coefficient value of water at another wavelength, and the absorbance value of the external absorber at the three wavelengths, A photoacoustic analysis method comprising: calculating a glucose concentration. A photoacoustic analysis method for compensating an influence of an internal characteristic of a tissue on a signal obtained by measuring an analyte including glucose in a subject tissue,
Irradiating the subject site with a near infrared laser pulse from a laser source in the subject site to generate a first ultrasonic pulse for each laser wavelength; and
Wherein during a period when the have a light absorbing material disposed between the subject region and the laser source so ultrasonic pulse is detected by the ultrasonic detector and propagates the tissue occurred within the light absorbing material Generating a second ultrasonic pulse in the target tissue using a laser pulse having the same laser wavelength and energy as in step (a);
The intensity ratio between the first ultrasonic pulse generated in step (a) and the second ultrasonic pulse generated in step (b) was calculated and generated in step (a). (C) generating a normalized analyte-related photoacoustic signal by compensating the measured value of the first ultrasonic pulse with the influence of the bulk tissue characteristic of the subject site;
Using the intensity ratio of the ultrasonic pulse, the absorption coefficient value of glucose at one wavelength, the absorption coefficient value of water at two other wavelengths, and the absorbance value of an external absorber at the three wavelengths, the tissue A photoacoustic analysis method comprising the step (d) of calculating a glucose concentration in the inside. A photoacoustic analyzer that non-invasively measures an analyte in a target tissue of a subject,
Means for generating laser pulses at multiple wavelengths;
Means for irradiating the target tissue with the laser pulse;
Means for detecting a photoacoustic signal from the target tissue excited by the laser pulse;
Means for disposing an external absorber between the irradiated portion of the laser pulse and the target tissue;
Means for generating an intensity ratio of a plurality of photoacoustic signals;
A photoacoustic analysis comprising an arithmetic means for calculating the concentration of the analyte from the intensity ratio of the photoacoustic signal, the optical parameter of the analyte in the target tissue, and the optical parameter of the external absorber. apparatus.

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