A non-invasive method for the measurement of body fluid analytes
The present invention relates to a method for the non-invasive measurement of analytes in body fluids. It further relates to an apparatus for the non-invasive meas- urement of the presence or absence of analytes in body fluids.
Background of the invention
The measurement of analytes, such as glucose in blood is traditionally carried out by extracting a blood sample from the body followed by the measurement of a particular analyte.
It is estimated that a disease like diabetes afflicts over 100 million people worldwide. By year 2010, 31 million people are expected to be afflicted with diabetes in Europe, and 220 million people worldwide. Frequent monitoring of blood glucose is proposed to prevent complications due to diabetes. Current blood glucose tests involve pricking a finger with a lancet. This method presents obvious disadvantages to the diabetic. It is therefore of the outmost importance that reproducible non-invasive methods for determining blood analytes, such as glucose are developed.
In recent years more sophisticated techniques for the measurement of body fluid analytes have evolved. These involve various optical techniques. Non-invasive optical measurement of a body fluid analyte can be performed by directing a beam of light onto the body. The light is modified by the tissue after transmission through the target area. The content of the tissue will be optically fingerprinted by the diffuse light that escapes the tissue it has penetrated. The absorbance of light by any tissue depends on the chemical components in the tissue.
The theoretical basis for non-invasive analyte monitoring is based upon quantitative vibrational spectroscopy, such as infrared spectroscopy. Infrared spectroscopy measures the electromagnetic radiation that a substance absorbs at various wavelengths. The atoms constituting the molecules of the tissue are not stationary, but vibrate constantly. Absorption of light of the appropriate energy causes the molecule to become excited to a higher vibrational state. The excitation of the molecule oc-
curs only at certain discrete energy levels, which are characteristic for a particular molecule.
Many analytes absorb at multiple frequencies in both the mid- and near infrared fre- quency regions. However, most fundamental vibrational absorptions occur in the mid-infrared frequency region. The absorption bands of these analytes are often close, sometimes even overlapping. Several biological molecules, such as glucose absorb in the mid-infrared spectrum. For example, glucose has characteristic fundamental absorbances at 1033 cm"1 (9.7 μm) and 1080 cm"1 (9.26 μm). This extinc- tion is due to the fundamental, or primary absorption of the glucose molecules. This means that the amount of light scattered back at the fundamental absorption wavelength is less than the light scattered back from any other frequency region. The harmonics and overtones of the molecular vibrations in glucose are in the 1400- 1600 cm"1 range.
Due to the many overlapping absorption bands of various analytes, such as glucose in the mid-infrared range, previous attempts to conduct measurements of analytes have been performed in the near-infrared region, where the measurements are directed toward the overtones of the molecular vibrations of the analytes.
Such is the case in for example, US 5,830,132 describing the non-invasive analyte monitoring of glucose levels in humans. The measurement is performed by transmitting light of infrared wavelength of 0.4-2.4 μm through a finger nail. The irradiation of the nail is chosen because the optical penetration through the nail is greater than the skin tissue. The method utilises either transmitted or back-scattered light from the blood carrying vessels, in the near-infrared spectrum. In the document it is described how the measurement of glucose is performed in the wavelength region of 0.4-2.4 μm, due to the fact that measurements in other regions are impossible because of the absorbance of water.
However, the penetration of light at these wavelength intervals typically is less than 100 μm. This penetration depth has proven to be impractical for measurements through the skin tissue, and therefore previously described techniques have not been successful due to the lack of light penetrating through the tissue.
To avoid having to penetrate multiple cell layers, some researchers have measured glucose in the aqueous humor of the eye. In the eye light travels through the cornea. The cornea is contrary to all other tissues translucent, and more importantly there is substantially no scattering of light in the cornea. The aqueous humor of the eye is a limped liquid characterised by having a content of urea and glucose lower than in the blood plasma.
Vibrational spectra of molecules can also be obtained through Raman scattering.
J. Lambert et al. (LEOS Newsletter, April, 1998) disclose a method for the in vitro measurement of glucose in a rabbit aqueous humor model using Raman spec- trometry. Raman spectrometry is used to determine the characteristics of a particular scattering material (the incident light differs in absorption band from that of the substance). J. Lambert et al. measured glucose levels by applying laser emitting light to the artificial aqueous humor. The Raman spectrum for principal analytes of the aqueous humor were determined. These analytes were glucose, lactate, ascor- bate, and urea. The results proposed the possibility of using Raman spectroscopy of the aqueous humor to the estimation of blood glucose and other analytes non- invasively.
In LEOS Newsletter (April, 1998) R. Tarr and P. G. Steffes disclose two different theoretical experimental set up systems for the non-invasive method for the measurement of D-glucose in the ocular aqueous humor using Raman spectroscopy. The system set ups are aided by a computer model for the generation of wave propaga- tion. The researchers conclude that the sensitivity of the systems need to be improved in order to measure the glucose level of the aqueous humor.
A major problem for spectral analysis in the mid-infrared range is the lack of suitable light sources. Recently, compact lead salt diode lasers at this particular wavelength have become available on the market. Unfortunately these lasers have to be cryo- genically cooled for operation. However, the latest laser development is quantum cascade (QC) lasers using conventional GaAs technology operating in the mid- infrared spectrum. Quantum cascade lasers are increasingly used in experimental mid-infrared optical systems, replacing lead-salt diode lasers. High power quantum cascade lasers operating at room temperature have been demonstrated by Namjou
et al. (Opt. Lett. 23, 219, 1998). A high power long wavelength quantum cascade laser operating at 11.5 μm above room temperature (320 K) has been demonstrated by Faist et al. (IEEE Photonics Tech. Lett. 10, 1100, 1998). High temperature continuous wave operation of 8 μm quantum cascade laser has been reported by Sil- vken et al. (Appl. Phys. Lett. 74, 173, 1999). Quantum cascade lasers that emit at multiple wavelengths have also been developed. (A. Tredicucci et al. Nature, 396, 350, 1998). The above developments all point to the fact that cheap and compact light sources at specific wavelengths will be available for home care use in the near future.
The present invention presents a method of non-invasive measurement of a body fluid analyte using infra red and Raman spectrometry in the mid-infrared range. The method is an improvement of the practical and frequent measurement of blood fluid analytes, and may be used by both private and professional individuals. The inven- tion discloses a method of analyte measurement through a complex multi-layered tissue, such as the skin tissue, as opposed to prior art measurements in areas of significant anatomical difference to the skin tissue, such as the aqueous humor.
Summary of the invention
The present invention relates to a method for the non-invasive measurement of the presence or absence of at least one animal, including human, body fluid analyte, comprising the steps of:
irradiating tissue with electromagnetic radiation having at least one wavelength of more than 2.5 microns,
detecting at least one parameter of the electromagnetic radiation reflected from the tissue, wherein the parameter is able to be correlated to the presence or absence of the at least one analyte,
correlating the at least one electromagnetic radiation parameter to the presence or absence of the at least one analyte.
Further the invention describes an apparatus for the non-invasive measurement of the presence or absence of at least one animal, including human, body fluid analyte, comprising the means for:
- irradiating tissue with electromagnetic radiation having at least one wavelength of more than 2.5 microns,
- detecting at least one parameter of the electromagnetic radiation reflected from the tissue, wherein the parameter is able to be correlated to the presence or ab- sence of the at least one analyte,
correlating the at least one electromagnetic radiation parameter to the presence or absence of the at least one analyte.
In another aspect the invention discloses the use of a method for the measurement of at least one animal, including human, body fluid analyte, and the use of an apparatus for the measurement of at least one animal, including human, body fluid analyte.
Drawings
Fig.1 shows the visible absorption spectrum of a fingernail. The absorption is seen to decrease almost exponentially as a function of wavelength. A theoretical fit to the measured curve as an exponential decay predicts a value of the absorbance of 1.5 at a wavelength of 10 microns. The theoretical fit is also shown in the figure.
Fig. 2 shows a confocal Raman spectrum of the tissue through a fingernail. The spectrum was recorded at a wavelength of 780 nm in order to avoid any excessive absorption or scattering at lower wavelengths. Many of the absorption features of the spectra can be correlated with those found by Williams et al. ("Raman spectra of human keratotic biopolymers: Skin tissue, Callus, Hair and Nail", A. C. Williams, H. G. M. Edwards and B. W. Barry, Journal of Raman Spectroscopy, vol. 25, 95-98
(1994)).
In Fig. 3 (1 ) is a mid-infrared laser. This can be a tunable laser covering the wavelength range 9.2 to 10.7 microns. The light from the laser is shone at the finger
through the fingernail, which is shown stylistically as (3). The laser passes through a beamsplitter (2), in order to gather the scattered light from the nail and the tissue below the nail. (4) is a detector to detect the scattered light and (5) is an electronic processing and display unit.
Fig. 4 is a modification of the set-up shown in Fig. 1. In this case, a polariser (6) is placed in the path of the incident beam and another polariser (7), whose direction of polarization is orthogonally oriented to (6). In this case, light is detected at the same wavelength. All the polarised light resulting from scattering from the nail can be eliminated by the polariser (7) and only depolarised scattering from the tissue and blood can be detected at the detector (4).
Fig. 5 is a simple set-up to detect Raman scattering from the nail and tissue. In this case, a filter (8) is placed in the scattered light such that it only transmits those frequencies that are Raman shifted by the glucose molecules.
Fig. 6 is the same as Fig. 2 except the polariser (7) is mounted on rotation stage. In this case, the rotation of the plane of incident radiation due to the glucose molecules can be detected. Suitable precautions have to be taken in this case in order to eliminate the plane of rotation by other biologically active molecules, such as keratin, cholesterol etc. In addition a magnetic field may be applied.
Detailed description of the invention
The object of the present invention is to provide a method and an apparatus for the non-invasive measurement of a body fluid analyte.
The present invention is based on the fact that absorbances of analytes of a body fluid may be accessible through the skin tissue. The method according to the invention for the non-invasive measurement of the presence or absence of at least one animal, including human, body fluid analyte, comprises the steps of:
irradiating tissue with electromagnetic radiation having at least one wavelength of more than 2.5 microns,
- detecting at least one parameter of the electromagnetic radiation reflected from the tissue, wherein the parameter is able to be correlated to the presence or absence of the at least one analyte,
- correlating the at least one electromagnetic radiation parameter to the presence or absence of the at least one analyte.
It is an object of the present invention to irradiate tissue with electromagnetic radiation having at least one wavelength of more than 2.5 microns. This range of wave- lengths of particular interest to the invention is in the mid-infrared spectrum.
It is an important aspect of the invention that the at least one wavelength corresponds to a wavelength outside the absorption range of the analyte and thereby provides a reference.
The wavelength applied is dependent on the analyte of interest. According to the invention in one embodiment the analyte may have an absorbance in the wavelength of irradiation of more than 5 microns. Yet, in another embodiment of the invention the wavelength of irradiation is more than 7 microns. In a further and pre- ferred embodiment of the invention the wavelength of irradiation is more than 9 microns. The absorbance spectrum of glucose is entailed in the latter wavelength range.
In one aspect of the invention the irradiation of the tissue is performed with at least one wavelength. In a further aspect of the invention the at least one wavelength corresponds to the absorption of an analyte.
It is also important that an absorption reference is made available and therefore the at least one wavelength corresponds to a wavelength outside the absorption range of the analyte and thereby provides a reference.
When a tissue is irradiated with a beam of light some will transmitted, some will be absorbed, and a small fraction will be scattered. The latter phenomenon is the basis for the Raman technique, which is an indirect measurement of the concentration of an analyte. In Raman spectrometry the tissue is irradiated with light and the scat-
tered light is examined through a spectrometer. Most of the scattered light consists of the parent line produced by absorption and reemission. Weaker lines occur at lower and higher energy and are due to absorption and reemission of light coupled with vibrational excitation or decay respectively. The difference in frequency be- tween the parent line and the Raman line is the frequency of the corresponding vibration.
According to the invention the tissue is irradiated for 1 to 60 seconds, such as from 1-30 seconds or 1-20 seconds, or 1-10 seconds, or 3-7 seconds. In a preferred em- bodiment the tissue is irradiated for approximately 5 seconds. The length of time of which the tissue is irradiated is dependent on the tissue type in question and of the nature of the analyte to be measured.
It is an object of the present invention to detect at least one parameter of the elec- tromagnetic radiation reflected from the tissue. The parameter detected may be correlated to the presence or absence of the at least one analyte.
According to the invention the parameters measured may be intensity, wavelength, and polarisation. Variations in intensity of the back scattered reflected light will be less due to scattering and absorption due to the constituent molecules in the tissue.
Furthermore, since part of the incident energy is absorbed, the scattered light may also occur at different wavelengths due to Raman scattering. A change in the polarisation of the scattered light as well as a rotation of the plane of the incident polarised light may also occur due to fibres aligned in a particular direction in the tissue or due to the chirality of the molecules constituting the tissue, or due to an external magnetic field.
It is an object of the present invention to irradiate any skin tissue, such as finger tissue. In the context of the invention the term "tissue" is meant surface tissue, such as the skin tissue, nails, or mucosa. It does not cover volumes of fluid, such as the aqueous humor of the eye, nor artificial aqueous humor.
In one embodiment of the invention the body analyte is measured through at least one finger. The light is shone on at least one finger, whereby the level of the desired analyte is determined.
According to the invention different areas of tissue may be irradiated for the measurement of different analytes. In one aspect of the invention at least two different areas on at least one finger are irradiated. This may provide for the simultanous measurement of at least two different analytes.
In yet another embodiment of the invention at least two different areas of tissue on the at least two different fingers are irradiated.
In a further embodiment at least one area of tissue on at least two different fingers are irradiated. This may serve to increase the significance of measurement of the level of the analyte.
However, in a preferred embodiment of the invention the irradiated tissue is at least one fingernail. Nails are composed of keratin fibres more or less aligned parallel to the direction of growth. The nail itself is translucent and colourless, allowing the colour of the blood in the superficial capillaries in the nail bed to show through. Thus, it is possible to access the blood capillaries and other body fluids through optical means relatively easy.
Furthermore, according to the invention the basis of at least one fingernail is irradiated. The basis of the fingernail is thinner than the top of the fingernail, and therefore radiation directed towards the fingernail basis must travel a shorter distance before reaching the analyte in question, and the back scattered light may represent a more precise starting point for the analysis of the levels of the analyte.
It is understood that the tissue fibres of the invention are substantially parallel. In the present context the term "substantially parallel" means that the fibres are aligned almost exclusively parallel. The alignment of the tissue fibres is important for the direction the radiation will pursue once it has contacted the tissue, and is scattered back. Accordingly, a preferred tissue area is an area where at least a part of the tissue comprises fibres substantially parallel.
It is an object of the invention to distinguish between the analyte of interest and other components, such as other analytes. This may be achieved by correlating the information obtained to known values and standards, and thereby correcting the value of interest.
In case of a nail the spectrum of keratin shows an absorption at 1087 cm"1 (corresponding to a wavelength of 9.2 microns). The refractive index of keratin (hair) is 1.555. Since the absorption wavelengths for keratin and other analytes, such as glucose are different, it is possible to distinguish between keratin and glucose at a wavelength of 9.7 μm because keratin does not absorb at 9.7 μm.
In one aspect of the invention the optical properties at a characteristic wavelength of keratin is measured. Furthermore, according to the invention this characteristic wavelength of keratin is 9.1-9.3 microns.
In a further aspect of the invention the optical properties at a characteristic wavelength of water is measured. This characteristic wavelength of water according to the invention is between 10.6-10.8 microns. Since the absorption due to water in the 9 - 1 1 μm range is known, an interpolation may be performed for water absorption at 9.7 μm.
In yet another aspect of the invention the characteristic wavelengths at which measurements are performed are those of the analyte, water and keratin. These characteristics are important to determine due to the high content of water in body fluids and the composition of a nail for example as described above.
Furthermore, it is an object of the present invention to provide for a method, wherein the incorrect physical positioning of the finger(s) when irradiated releases an alarm signal. For the purpose of the invention the finger(s) is placed in a device designed to accommodate the finger prior to irradiation. However, if the finger is not positioned correctly inside the device an alarm will go off. This may enable the person carrying out the measurement and may also ensure the correct measurement of the analyte in question.
The analytes measured may be any body fluid analyte, such as glucose, urea, cholesterol, and alcohol.
It is envisioned that the method of the invention may be applied to fields, wherein a need for the uncomplicated and fast determination of a body fluid analyte is impor- tant. As mentioned above one application may be the determination of blood glu-
cose, for example in patients with diabetes. Another application may be in the general health care system for the determination of analytes, such as narcotics. This application may result in the rapid diagnose of patients who have overdosed. A further application may be for the diagnostics of anaemia or cancer.
In another aspect of the present invention the analyte is alcohol whose absorption lies between 6 and 16 μm. The invention measuring alcohol may be useful in law enforcement situations, such as when the instant determination of the blood alcohol percentage of motor vehicle drivers is required.
Without being bound by theory the present invention may be applied to determine a particular body fluid analyte in a transgene animal. The transgene animal may have been manipulated in order to express a particular analyte, such as a protein. The present invention provides for a method of determining the presence or absence of a particular analyte.
In a preferred embodiment of the invention the analyte is glucose absorbing at a wavelength of between 6 and 14 microns and more specifically a wavelength of 9.6- 9.8 microns.
Further to the invention the analyte is urea having a wavelength of 6.8 microns. The determination of the concentration of urea is important for diagnostic purposes, such as in certain diseases.
In another aspect of the invention the analyte is cholesterol absorbing at a wavelength of between 6.5 and 10.5 microns, and having a wavelength of 7.3 microns. The application of the present invention for the measurement of cholesterol may be beneficial in self examination situations, wherein a person wishes to obtain information on the status of the cholesterol levels.
The scope of the invention is not limited to analytes or applications mentioned herein, but may be any analytes or applications of interest.
According to the invention the analyte may be present in any body fluid, such as serum and plasma. In a preferred embodiment of the invention the body fluid is
blood. Many important analytes of interest for diagnostic purposes are present in the blood. In addition to holding vital analytes, the blood capillaries are vastly distributed in the body, some being in close proximity to the surface of the body, and thereby being accessible for irradiation.
In the event that the pigmentation of the irradiated tissue is of importance to the quality of the measurement, tissue of even pigmentation is preferred for the irradiation. The pigmentation of the tissue may be considered when analysing the data of measurement.
According to the invention the presence or absence of the at least one body fluid analyte is measured quantitatively, by determining the amount of a given analyte. Raman spectrometry may be one example of a quantitative analysis. Infrared spec- trometry is another example of quantitative analysis.
In another aspect of the invention the presence or absence of the at least one body fluid analyte is measured qualitatively. In many cases, it is enough just to determine whether a particular species is present in the tissue. In this case, a positive signal such as a signal at a particular wavelength is enough to show the presence of the species.
In another aspect of the invention the temperature of the tissue is measured. The temperature of the tissue may be measured prior to the irradiation or it may occur simultaneous to the irradiation. Additionally, the temperature may be measured after the irradiation of the tissue. Regardless of the time of the temperature measurement the temperature is considered when determining the absence and presence of the at least one analyte.
The measurement of the temperature is important due to individual differences in temperature. For example the skin tissue temperature may vary greatly from individual to individual, and it is also dependent on the environment. It is mostly in the external body tissue layers that fluctuations occur. According to the invention the temperature differences may be compensated for.
The method according to any of the preceding claims of calculating the analyte concentration by compensating for variations in the intensity.
The method according to any of the preceding claims of calculating the analyte con- centration by measuring the back scattered electromagnetic radiation from tissue components other than the analyte(s).
To obtain a clear and concise image of a band of an analyte it may be necessary to use filters. In one aspect of the present invention filters are used for the measure- ment at shifted wavelength.
More particularly, in one embodiment of the invention, interference filters are used for the isolation of a band of wavelengths. In another embodiment of the invention, notch filters are used for the isolation of a band of wavelengths.
The filters used according to the invention may be any filter capable of filtering "noise" interfering with the detection of the band of the analyte measured.
Another object of the present invention is to provide for an apparatus for the non- invasive measurement of the presence or absence of at least one animal, including human, body fluid analyte, comprising the means for:
irradiating tissue with electromagnetic radiation having at least one wavelength of more than 2.5 microns,
detecting at least one parameter of the electromagnetic radiation reflected from the tissue, wherein the parameter is able to be correlated to the presence or absence of the at least one analyte,
- correlating the at least one electromagnetic radiation parameter to the presence or absence of the at least one analyte.
In a preferred embodiment the apparatus according to the invention comprises:
- a electromagnetic radiation source with a tunable laser, and
at least one detector, and
at least one beamsplitter, and
- at least one electronic processing and display unit.
As previously mentioned a major problem for spectral analysis in the mid-infrared range is the lack of suitable light sources. The preferred source of light according to the invention is a laser, such as a compact diode laser or a quantum cascade laser as mentioned above.
Since the measurements of the invention are in the mid-infrared area, the apparatus according to the invention, has at least one detector which is a mid-infrared detector. The apparatus may entail more than one detector depending on the nature of the components of the apparatus
In a preferred embodiment of the invention the at least one detector of the apparatus is a HgCdTe detector.
In the apparatus of the invention the optical path length through the tissue may be less than 15 mm. By the term "optical path length" is meant the distance travelled by light through the tissue. There is no limit on the distance between the source and the detector. In one embodiment of the invention, the source and the detector may be placed directly on the tissue in question.
In one embodiment of the apparatus according to the invention, the electromagnetic radiation from a laser is shone at the tissue through a beamsplitter, and then detected, followed by electronic processing.
In another embodiment of the invention the apparatus optionally comprises at least one polarising filter in addition to the above mentioned components of the apparatus. According to the invention, a first polariser is placed in the path of the laser electromagnetic radiation beam and a second polariser is placed orthogonally oriented to the first polariser, and further a filter is placed in the path of the scattered electromagnetic radiation.
A major problem of the detection of analytes, such as glucose using spectral analysis in the mid-infrared range is that it is obscured by water. Water is a critical matrix component in that its absorption of light creates strong absorbance bands. This can be corrected for through a measurement of the back scattered light just outside the absorption of the analyte band, and from known absorbance tables in the literature.
The corrected absorption spectrum at 9.7 μm may resemble a correlation to a particular analyte concentration level in the blood, such as the glucose concentration level.
In another aspect of the invention the apparatus may be applied with a magnetic field. Such a magnetic field may be applied to enhance the optical rotation.
In addition to the natural rotation of the plane of polarization, glucose molecules also show a magnetic optical rotatory effect. According to the invention a magnetic field is applied parallel to the direction of the incident radiation. In order to distinguish this effect from the natural rotation of the plane of polarization, the magnetic field is modulated at a few hertz, and a lock-in detection mechanism may be employed. This effect may be enhanced when the irradiated light has a wavelength close to an absorption maximum of the analyte. This absorption can be due to the fundamental or harmonic frequencies of the analyte, and thus may also be measured in the near- infrared spectrum. Further, the magnetic field may be applied perpendicular to the light path.
In a another aspect the present invention relates to the use of a method as defined above for the measurement of at least one animal, including human, body fluid analyte, and the use of an apparatus for the measurement of at least one animal, including human, body fluid analyte.
The following are examples of the experimental set-ups of the invention. The ex- pehments are illustrated by the Figures 1-3. The figures illustrate the experimental set ups when determining the concentration of blood glucose through a finger nail.
Experimentals
Example 1
Figure 1 is an illustration of the measurement of the back-scattered (reflected) intensity of light at 9.7 μm through the nail. A reference measurement of the scattered light from the nail alone is made first at 9.2 μm, 9.7 μm and 10.7 μm, respectively. Then measurements are made through the nail from the blood capillaries at the same wavelengths. Measurements at 9.2 μm and 10.7 μm serve as reference measurements at wavelengths outside the glucose absorption band, by making use of the tunability of the laser, to eliminate interference due to keratin and water. Since the absorption due to water in the 9 - 11 μm range is known, an interpolation is performed for water absorption at 9.7 μm. The corrected absorption spectrum at 9.7 μm resembles a correlation with the glucose concentration level in the blood.
Example 2
Figure 2 shows another embodiment of the invention. In this case, the polarization properties of the aligned keratin fibres in the finger nail are utilised to eliminate the back scattered radiation from the nail. Since the keratin molecules grow along the length of the nail, the back scattered light will be polarised. This is eliminated by using another polariser oriented at 90° to the first, crossing out polarised light.
Example 3
Figure 3 displays another embodiment of the invention. In this case, Raman scattering from glucose molecules will be utilised for the detection. Raman scattering occurs when molecular vibrations absorb part of the incident light and emit the rest as longer wavelength irradiation. In this case, through the utilisation of a filter specifically designed for the Raman shifted frequencies, enhancement in the sensitivity is achieved.
Example 4
Yet another embodiment is shown in Figure 4. Here, the rotation of the plane of po- larisation associated with glucose molecules can be utilised. Again, by making ref-
erence measurements from the nail alone and at different wavelengths, the interfering effects due to other molecules is eliminated.