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BACKGROUND OF THE INVENTION
1. Field of the invention
The present invention relates to a spectroscopic method for measuring the
concentrations of sugars such as glucose, saccharose, fructose and the
like and, more particularly, to a method and apparatus for measuring sugar
concentrations in foods, fruits and other agricultural products, and the
like and in body fluids of man, animals, and other creatures by a
non-invasive or non-destructive and easily repeatable technique, that is,
without taking the sugar medium as sample for measurement out of the
object of examination.
2. Related art
A non-invasive and easily repeatable measuring method as mentioned above is
specially useful in that the method dispenses with the step of taking a
sample, for example the body fluid, out of the object of examination in
the measurement procedure. In this method, however, it is unsatisfactory
that the regions amenable to the measurement should be limited to the
outermost layer of the object of examination. For example, in a fruit as
an object of such examination, the pericarp which constitutes the
outermost layer is different from the sarcocarp in the interior of the
fruit with respect to the structure of the tissue, and the composition,
the related distribution, and the like of the chemical component. The
determination of the sugar concentration in the sarcocarp necessitates
relevant measurement at deeper positions than the outermost layer.
In U.S. Pat. No. 4,655,225 is described a prior art technique whereby
sugars in human serum, especially glucose therein, can be quantitatively
determined non-invasively. This technique, known as "incident angle
modulation method", is a spectroscopic method wherein, in principle, a
sample is irradiated with light beams from the outside and the light beams
diffused and reflected from within the sample are spectroscopically
analyzed. In this method the angle of incidence which light waves makes
with a sample is changed. When this angle is small, the light beams
penetrate deep underneath the outermost layer, and when the angle is
large, the depth to which the light beam penetrates decreases. Therefore,
by changing the depth to which a light beam penetrates underneath the
outermost layer so as to find the respective spectral signals from the
different depths, information from a deeper point, that is, the sugar
concentration, can be discretely determined on the basis of the
differential signal therebetween.
However, the above-mentioned prior art technique when practiced raises a
problem in that the mechanism for modulating the angle of incidence is
complicated on the one hand and the change in the incidence angle of light
beam impairs the reproducibility under the influence of a resultant change
in the reflection characteristic at the surface of the outer layer.
In said prior art method the light beam used for measurement are of
wavelengths in the near infrared region, which are 2,270.+-.15 nm,
2,100.+-.15 nm, 1,765.+-.15 mn, and 1,575.+-.15 nm, and the reference
wavelengths used are in the range of 1,000 to 2,700 nm. In order to
enhance the accuracy of the measurement, however, shorter wavelengths in
the near infrared region should be used for the measurement. The reason is
that the liquid in a living body, especially in a fruit, agricultural
product, or the like, consists of water in such a large proportion that
the optical penetration depth of light beams to water, which assumes a
larger degree on the side of shorter wavelengths, is of importance in the
practice.
Wavelengths in the intermediate infrared range, especially those in the
range of 7,500 to 15,000 nm called "fingerprint region", are effective in
spectral analyses and have long since been used for identification and
determination of organic compounds. Since, however, the water existing as
a background exhibits such a high rate of absorption for said wavelength
region that it is generally considered impractical to determine a specific
component combined with water by the fingerprint region. Although light
beams in visible region have a good optical penetrability in water, there
exists no spectrum of a characteristic absorption bands for sugar in the
visible light region.
SUMMARY OF THE INVENTION
Accordingly, an essential object of the present invention is to provide a
spectroscopic measuring method, according to which the measuring apparatus
can be made simpler than in the prior art and by using light beam of
shorter wavelengths in the near infrared region the accuracy of the
measurement can be enhanced.
Another object of the present invention is to provide an apparatus which is
adapted to the practice of the above-mentioned method of this invention.
In accomplishing the above-mentioned objects there is provided, according
to the present invention, a spectroscopic method for measuring the
concentrations of sugars in samples, which comprises a first step wherein
the light intensity of a light source in an interferometer is set at a
first prescribed value and the outcoming light beam from said
interferometer irradiate a sample placed in an integrating sphere, a
second step wherein the diffusely reflected light from said sample
collected by an integrating sphere is detected by a photo detector, a
third step wherein the absorption spectrum of the sugar under measurement
at a first depth in said sample is determined by processing the electrical
signal from said photo detector by computer, a fourth step wherein the
light intensity of the light source in said interferometer is set at a
second prescribed value and the absorption spectrum of the sugar under
measurement at a second depth in said sample is determined in the same
manner as in said first, second and third steps, and a fifth step wherein
the absorption spectrum of the sugar under measurement at a deeper region
between said first and second depths in the sample is determined on the
basis of the difference between the respective absorption spectrum
determined in said third and fourth steps so that the concentration of the
sugar under measurement in the deeper region in said sample can be
determined on the basis of said absorption spectrum at the deeper region
between said first and second depths, the wavelengths for the use for said
absorption spectrum being selected from a wavelength band of from 950 to
1,150 nm, from 1,150 to 1,300 nm, or from 1,300 to 1,450 nm.
Since, according to the above-mentioned measuring method, the wavelengths
selected for absorption spectrum are shorter and closer to the visible
region than in the prior art methods, the light beam can penetrate deeper
in the living tissue, and therefore, the region in the body tissue
amenable to the measurement is widened beyond that in the prior art. Where
the method of the present invention is used to determine the
concentrations of sugars in the bloodstream in a human body, the selection
of wavelengths from the wavelength bands specified above for the light
beam enables them to reach the corium where capillaries are spread or to a
point sufficiently close to the corium so that the measurement can be
carried out non-invasively, that is, without taking out the sample blood.
Another advantage of this method is that the selection of wavelengths as
specified above, that is, the use of shorter wavelengths which are closer
to the visible region, for the light-throughput is increased, so that the
efficiency of the apparatus can be improved and the accuracy of the
measurement can be enhanced.
A further advantage of this method is that, since a technique of changing
the light intensity of the light source is introduced so as to eliminate
the background noise at the epidermal layer of the sample in measuring the
concentrations of sugar at a depth beneath it as desired, the practice
dispenses with a complicated apparatus such as a light beam incident angle
modulator which was required in the prior art.
In accordance with the present invention, there is also provided a
spectrophotometric apparatus for measuring the concentrations of sugar in
samples, which comprises an interferometer wherein superposed light beams
are emitted, a controller for adjusting the light intensity of the light
source which is placed in said interferometer, an integrating sphere to
collect diffusely reflected light from a sample placed at said integrating
sphere, a photo detector for detecting said collected light and converting
it into an electric signal, a processing means for processing the electric
signal from said photo detector so as to compute the absorption spectrum
of the sugar in said sample to be determined and for determining the
concentration of the sugar under measurement on the basis of said
absorption spectrum, and a means of selecting wavelengths for said
absorption spectrum from a wavelength band of from 950 to 1,150 nm, from
1,150 to 1,300 nm, or from 1,300 to 1,450 nm. The processing means
includes a means whereby, on the basis of the difference between the
absorption spectrum of the sugar under measurement at a first depth in
said sample which is computed for the light intensity of the light source
in said interferometer set at a first prescribed value and the absorption
spectrum of said sugar at a second depth which is computed for the light
intensity of said source set at a second prescribed value, the absorption
spectrum of the sugar under measurement at a deeper region between said
first and second depths in said sample is computed and includes
furthermore a means whereby, on the basis of said absorption spectrum at
the deeper region between said first and second depths, the concentration
of the sugar under measurement in the deeper region in the sample is
computed.
BRIEF DESCRIPTION OF THE DRAWINGS
These and other objects and feature of the present invention will become
apparent from the following description taken in conjunction with the
preferred embodiment thereof with reference to the accompanying drawings,
in which:
FIG. 1 schematically illustrates an apparatus used in the practice of the
measuring method of the present invention;
FIG. 2 shows the extinction coefficients of water in the near infrared
region;
FIG. 3 shows absorbance spectrum of purified water, an aqueous solution of
glucose, and an aqueous solution of saccharose;
FIG. 4 shows differential spectrum between an aqueous solution of glucose
and pure water;
FIG. 5 shows the differential absorbance with respect to differential
spectrum between an aqueous solution of saccharose and pure water;
FIG. 6 shows differential absorbance between pseudo-pure glucose spectrum
and pseudo-pure saccharose spectrum;
FIG. 7 shows the differential absorbance with respect to the differential
spectrum between pseudo-pure glucose spectrum and pseudo-pure saccharose
spectrum;
FIG. 8 shows the concentrations of glucose determined according to the
invention, in relation to the actual measured concentrations, in the test
using aqueous mixtures of glucose and saccharose wherein specific selected
wavelengths were applied according to a model formula for determining
glucose concentrations as described in the example that follows;
FIG. 9 shows the concentrations of saccharose determined according to the
invention, in relation to the actual measured concentrations, in the test
using aqueous mixtures of glucose and saccharose wherein a model formula
for determining saccharose concentrations was used as described in the
example that follows; and
FIGS. 10 and 11 are the same representations as FIGS. 8 and 9 respectively,
except that certain selected optimum wavelengths other than those
specified in this invention were used.
THE DETAILED DESCRIPTION OF THE INVENTION
The present invention will now be described in detail hereunder with
reference to the accompanying drawings.
Referring to FIG. 1, there is illustrated schematically an apparatus used
in the practice of a measuring method embodying the present invention,
which is as follows.
The illustration shows a Michelson type interferometer denoted by MI, which
comprises a light source 2, a collimator 7a, i.e. a spherical reflecting
mirror, which receives the light from said source 2 and reflects the
parallel light beams, a beam splitter 4 that divides the collimated light
beam into two beams R.sub.1 and R.sub.2 which makes a right angle to each
other, one beam directed onto a fixed mirror 3 and the other onto a
movable mirror 5. The beams reflected by said fixed mirror 3 and movable
mirror 5 are recombined at said beam splitter 4. A movable mirror driving
device 6 is arranged so as to move said movable mirror 5 to and fro. Here
constructive and destructive interference occurs, depending on the
position of the moving mirror 5 relative to the fixed mirror 3. The light
intensity of the light source 2 can be adjusted by a light source
intensity controller 1.
The outcoming light beam emitted from the Michelson type interferometer MI
passes through a band-pass filter 13 which permits passage of the light
beam of predetermined wavelength band. The light beams of a selected
wavelength band are directed onto a spherical mirror 8 and reflected
thereby into an integrating sphere 9 through the inlet provided therein so
as to be focused onto a sample 15 placed at a window provided in the
integrating sphere. The light beam penetrates the sample and is then
diffusely reflected by the tissue underneath the epidermis and rise to the
surface. The reflected light beam from the sample is collected by the
integrating sphere and led to a detector 10, which is a photoelectric
converter, placed at another window provided in the integrating sphere.
The electrical signal produced by the detector 10 is amplified by an
amplifier 11 and converted by an A/D converter into a digital signal,
which is inputted to a computer 12. The signal, seen by the detector as a
movable mirror scans to and fro, is the interfrogram.
A detailed explanation will now follow hereunder with respect to the
wavelength region for the measurement which are selected by the band-pass
filter 13.
As touched upon already, in the objects of measurement in the practice of
the present invention, which are foods, fruits and other agricultural
products, man, animals, and other creatures, the liquid in a living body
subjected to the measurement consists of water in such a high proportion
that it is essential to consider the absorption of light beam in water in
the study of the application of the present invention.
In FIG. 2 are shown extinction coefficients of water in the near infrared
region. The extinction coefficients are shown along the axis of ordinate
in relation to wavelengths taken on the axis of abscissas. As is clearly
seen, the absorption of pure water is dependent upon the wavelength and
have peaks around 1.93 .mu.m, 1.43 .mu.m, 1.15 .mu.m, and 0.96 .mu.m. And
the depth of optical penetration to water is greater where the wavelength
is shorter. The absorbance of water at a wavelength of 1.93 .mu.m differs
from that at 0.96 .mu.m by about three to four orders of magnitude.
Therefore, if a wavelength band is capable of determining the chemical
component under examination, that is, the sugar, exists in the near
infrared region closer to the visible region than the bands for
measurement in the prior art, the use of the band enables the light beam
to penetrate deeper into a water-containing living tissue and reach a
point so deep as to show the chemical component at the value
representative of the concentration at the region under investigation. It
constitutes another advantage that the use of a wavelength band close to
the visible region enhances the light-throughput and, therefore, results
in improvement in efficiency of the measuring apparatus.
The present inventors, therefore, examined glucose and saccharose with
respect to the feasibility of quantitatively determining each of them
individually using wavelengths in the near infrared region, each of the
two kinds of sugars closely resembling the other in molecular structure
and their test as samples of sugars being of practical value. As a result,
it was discovered that, although near infrared spectrum of glucose
resemble those of saccharose so closely that spectrum of their mixture
overlap and interfere, there existed bands of shorter wavelengths than in
the prior art, with significant differences between the bands. Said
band-pass filter 13 is designed to pass only the light beam in these
specific wavelength bands.
A detailed description will follow hereunder with respect to light beam or
spectrum in these significant bands.
When samples were aqueous solutions, as shown in FIG. 3, it was difficult
to visually distinguish a significant difference in spectra between pure
water and an aqueous solution of saccharose or an aqueous solution of
glucose. The inventors then examined the differential spectrum between an
aqueous solution of glucose and pure water (see FIG. 4) and those between
an aqueous solution of saccharose and pure water (see FIG. 5). A
differential spectrum herein means what remains after subtracting from a
spectrum of an aqueous glucose or one of an aqueous saccharose the
spectrum of pure water equivalent in quantity to the aqueous constituent
of the aqueous glucose or the aqueous saccharose (this differential
spectrum will hereinafter be referred to as a spectrum of pseudo-pure
glucose or a spectrum of pseudo-pure saccharose). In FIGS. 4 and 5 the
number assigned to each curve refers to the concentration of glucose or
saccharose as follows:
No. 1: 2.0 Mol/l
No. 2: 1.0 Mol/l
No. 3: 0.5 Mol/l
No. 4: 0.25 Mol/l
No. 5: 0.125 Mol/l
Then, to find the differences between the respective spectrum of glucose
and saccharose, the spectrum of pseudo-pure glucose were compared with
those of pseudo-pure saccharose, as shown in FIG. 6. The resultant
differential spectrum (=pseudo-pure glucose spectrum - pseudo-pure
saccharose spectrum) are shown in FIG. 7. As mentioned already, glucose
and saccharose closely resemble each other in molecular structure and this
fact is reflected in their spectrum, as shown in FIG. 6. Despite this
similarity in molecular structure, however, significant difference can be
observed in the wavelength bands of from 1,150 to 1,300 nm and from 950 to
1,150 nm.
In a spectroscopic analysis of a three-component mixture consisting of
glucose, saccharose and pure water, can be determined by introducing a
mathematical formula using individually three specific wavelengths in said
1,150 to 1,300 nm band with significant differences observable therein,
which are 1,230 nm at which the absorbance of glucose is conspicuous and
relatively predominant, 1,205 nm at which the absorbance of saccharose is
conspicuous and relatively predominant, and a base wavelength of 1,285 nm
having no relevance to the concentrations of glucose and saccharose. By
applying this method unknown samples were determined, FIGS. 8 and 9
showing the results. FIG. 8 relates to an aqueous solution mixture of
glucose and saccharose and shows the concentrations determined by using a
model formula for determining glucose concentrations, said determined
concentrations shown along the axis of ordinate in relation to the actual
measured concentrations (known concentrations) taken on the axis of
abscissas.
The spectroscopic determination of the concentrations of components in a
chemical system is based upon the Beer-Lambert law which states that the
absorbance A is proportional to the concentration C (A=KC (K is a
constant)). Even if it is assumed that the Beer-Lambert law can be
expanded and applied to multi-component system, it is impossible to form a
model formula or compute therewith unless all possible sets of components
in the mixtures are specified. It is generally impracticable to prepare
pure standard samples by specifying all possible sets of components
especially in complex mixtures such as a living body.
On the other hand, the application of the inverse Beer-Lambert law (C=PA (C
is a constant)), wherein the concentration C is regarded as a function of
the absorbance A, enables a model to be formed and used in the computation
in relation to specific components, that is, without the need of
specifying all possible sets of components in a system. In other words,
for example, a model formula can be formed for practical computation with
respect to the concentration of glucose in a multicomponent system.
A model formula will be explained hereunder using a linear combination
comprising, for example, absorbances at three different wavelengths.
##EQU1##
where A(.lambda.1), A(.lambda.2), and A(.lambda.3) are the absorbances at
selected wavelengths of .lambda.1, .lambda.2, and .lambda.3;
P1, P2, and P3 are proportional constants for the absorbances A(.lambda.i);
Po is an intercept term of the model.
The absorbance A(.lambda.i) at the selected optimum specific wavelength
.lambda.i is divided into a component absorbance Ag(.lambda.i) depending
on the concentration of glucose and a component absorbance Ab(.lambda.i)
depending on the components other than glucose in the system.
A(.lambda.i)=Ag(.lambda.i)+Ab(.lambda.i) (2)
Ab(.lambda.i) includes, besides the contribution from the sugar components
other than glucose, the contribution from the background.
Then, by applying the equation (2) the equation (1) can be developed as
follows:
##EQU2##
where Cg is a term depending on the glucose component and represents the
true concentration of glucose; Cb represents the contribution from the
components other than glucose and that from the background. Then, most
optimum wavelengths .lambda.1, .lambda.2, and .lambda.3, optimum
coefficients P1, P2, and P3, and Po are determined by multiregression in
such a way as to satisfy
Cb=P1Ab(.lambda.1)+P2Ab(.lambda.2)+P3Ab(.lambda.3)+Pbo.fwdarw.0 (6)
By thus canceling the contribution from the components other than glucose
and that from the background, Cg representing the true concentration of
glucose under investigation becomes obtainable in the model formula.
Since the equation (4) becomes almost equal to the equation (1), the
concentration of glucose can be obtained by measuring the absorbances
A(.lambda.1), A(.lambda.2), and A(.lambda.3) at selected optimum
wavelengths of .lambda.1, .lambda.2, and .lambda.3. Since the
concentration of glucose is under investigation, the equation (1) is
remodeled into
Cg=Pg1A(.lambda.1)+Pg2A(.lambda.2)+Pg3A(.lambda.3)+Pgo (7)
The concentrations of the components other than glucose can be obtained in
a manner similar to the above procedure.
A linear combination of absorbances at three wavelengths has been dealt
with in explaining the model formula, but, without restricting the
practice of this invention to this particular type of formulas, a model
formula using absorbances at four wavelengths or more, doublet, or
cross-product absorbance terms can be formed so as to be adapted to the
accuracy required or particular cases of application.
An explanation will be given next with respect to the results of analysis
obtained by using the equation (7) as a model formula.
Three selected specific optimum wavelengths in the band of 1,150-1,300 nm
are .lambda.1=1,205 nm, .lambda.2=1,230 nm, and .lambda.3=1,285 nm. In
FIG. 8, wherein the concentrations determined according to the invention
are shown in relation to the actual measured concentrations, the straight
line is the 45 degree on which all the points should lie in the absence of
error. The marks "o" plotted therein, each representing the results
obtained relating to a certain number of samples, lie along the straight
line and shows that glucose can be individually determined by the use of
said selected specific optimum wavelengths.
In a manner similar to the procedure for glucose mentioned above,
saccharose can also be individually determined by using the three specific
optimum wavelengths (1,205 nm, 1,230 nm, and 1,285 nm). FIG. 9 shows, with
respect to aqueous mixtures of glucose and saccharose, the concentrations
determined by using a model formula for determining concentrations of
saccharose along the axis of ordinate in relation to the actual measured
concentrations (previously known concentrations) taken on the axis of
abscissas. The results obtained with respect to each sample lie around the
straight line on which all the points should lie in the absence of error.
Similarly, glucose, saccharose and pure water in a three component system
can each be individually determined by the use of three optimum
wavelengths (950 nm, 980 nm, and 1,107 nm) in the band of from 950 to
1,150 nm where significant differences are shown.
Also in the band of from 1,300 to 1,450 nm significant differences are seen
in spectrum between glucose and saccharose (FIG. 7 shows this phenomenon
only in part). Therefore, by using three optimum wavelengths (1,349 nm,
1,385 nm, and 1,400 nm) in this band each of said components can be
separated in spectrum. In a manner similar to the above-mentioned example,
analytical graphs were obtained mathematically with respect to unknown
samples. The results in FIG. 10 (concentrations of glucose) and FIG. 11
(concentrations of saccharose) show better linearity in the relationship
between the determined concentrations and the actual measured
concentrations.
The wavelengths for the light beam used in the measuring apparatus in FIG.
1 are selected from any of the three wavelength bands, i.e., 950-1,150 nm,
1,150-1,300 nm, and 1,300-1,450 nm, as mentioned above.
Referring to FIG. 1, a detailed description will now follow with respect to
the method of measurement of the concentrations of sugars in the tissue,
particularly those of glucose as an example, at the depth b of the sample
15.
The first step begins with adjusting the light from the light source 2 to a
prescribed intensity by means of the light source intensity controller 1
so as to examine the spectrum of the tissue at the outermost layer a of
the sample. In other words, the light beams irradiating the sample 15 is
so adjusted as to enter only the outermost layer a and be reflected back
toward the outside. The spectrum pertaining to the outermost layer a of
the sample 15 is then measured. The band-pass filter 13 then permits
passage of the light beam of the necessary wavelength band, for example,
in the range of 1,150-1,300 nm.
In the second step, the intensity of the light from the source is increased
by the light source intensity controller 1 so as to enable the light beam
from the interferometer MI sphere 9 to penetrate the sample 15 deeper to a
depth b. In this way the spectrum of the sample 15 pertaining to both the
outermost layer a and the deeper layer b are computed. For this deeper
layer b, too, the computer 12 executes a program similar to that for the
outermost layer a for the measurement.
In the third step, the computer 12 first computes the differential spectrum
between the two spectrum obtained at the first step and the second step,
secondly selects a number of wavelengths necessary for determining the
concentration of the component under investigation, that is, the glucose,
from said wavelength band of 1,150-1,300 nm, for example, three
wavelengths of 1,205 nm, 1,230 nm, and 1,285 nm therefrom, and thirdly
determines the concentration of glucose by introducing these selected
wavelengths into a prescribed mathematical equation as hereinbefore
described.
By following the above-mentioned spectrum procedure the background from the
outermost layer a where glucose is not at all present or not contained to
any significant degree can be eliminated and the concentration of glucose
in the tissue at a deeper point b can be determined.
Although the present invention has been fully described by way of example
with reference to the accompanying drawings, it is to be noted, here, that
various changes and modifications will be apparent to those skilled in the
art. Therefore, unless otherwise such changes and modifications depart
from the scope of the present invention, they should be construed as
included therein.
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