the invention provides two general methods of skin thickness prediction on the basis of near - ir ( nir ) spectral measurements . the first method also yields information relating to the structure and composition of the absorbing and scattering species in each layer . further , knowledge of the thickness and optical properties of the individual tissue layers can be applied in a method of pathlength normalization to minimize the interference due to the variation of the individual layers . the primary method takes advantage of the presence of key indicators . key indicators are the chemical or structural components that are primary absorbers and / or scatterers in each particular tissue layer , and that are not present in significant amounts ( spectrally ) in other layers . this allows for the exploitation of distinct spectral characteristics and features that are specific to certain tissue regions , or layers , based solely on such spectral measurements . the spectral manifestation of these key indicators makes it possible to quantify the primary constituents and to determine the thickness of the individual tissue layers . the key indicators are determined from a priori knowledge of the composition and structure of skin tissue layers . examples of key indicators are provided in the table below : for example , because water is present in the dermis in greater concentration than in the epidermis or subcutaneous tissue , water is specified as a key indicator for the dermis . similarly , because high concentrations of triglycerides are found primarily in adipose tissue with relatively little found in the epidermis or dermis , trigylcerides are specified as a key indicator for adipose tissue , also known as subcutaneous tissue . collagen bundles can be used as an additional key indicator for the dermis . the epidermis can be discriminated by the scattering and / or absorbance of keratinocytes , while the stratum corneum is distinguished by the scattering and absorbance of corneocytes , keratinized cells , and specialized lipids . the procedure for measuring the magnitude of the key indicators and skin thickness is shown in fig1 . first , a library of normalized nir absorbance spectra 10 of the key indicators is provided . the spectra 10 of the key indicators are stored in the memory of a computer associated with a spectrometer device . a suitable system for executing the procedures and methods disclosed herein is described in a related application to the current application , t . ruchti , s . malin an intelligent system for noninvasive blood analyte prediction , u . s . patent application ser . no . 09 / 359 , 191 , filed jul . 22 , 1999 . a nir absorbance measurement 11 of the targeted tissue site is made in the wavelength region ( s ) in which both the key indicators specific to the target layer absorb or scatter and in which light penetration to the target tissue layer is optimal . the normalized pure component spectra of the key indicators are projected 12 onto the measured absorbance spectrum . alternately , the spectra of the key indicators are used as a basis set and the method of partial least squares is used to determine the optimal magnitude of each to represent the measured absorbance spectrum . the calculated magnitude 13 of each normalized key indicator provides a relative concentration of its respective constituent in the tissue . a composition calibration model 14 is applied to the calculated magnitudes to determine the actual concentration 15 of the constituent . in the related application cited above ser . no . 09 / 359 , 191 , a detailed description of a procedure for calculating such a calibration model is given . alternatively , the relative concentrations of the key indicators are processed by an alternate calibration model 16 for estimating skin thickness to determine the thickness of the target layer 17 . it will be apparent to one skilled in the art that since key indicators are specific to a given layer , their relative absorbances are directly related to the thickness of the targeted layer ( s ). one skilled in the art will further appreciate that an overall thickness estimate may be arrived at by a simple summing of the thickness estimates of the individual layers . the skin thickness calibration model 16 is calculated from a calibration set ( not shown ) of exemplary measurements that provides both the relative concentrations of the key indicators , calculated from absorbance spectra , and the thickness of each tissue layer . the calibration model is determined through multiple linear regression , partial least squares regression , artificial neural networks or other techniques such that the thickness of each layer is predicted through a mathematical mapping of the relative magnitude of the marker constituents . the related application ser . no . 09 / 359 , 191 , previously referred to , provides a detailed description of a procedure for calculating the skin thickness calibration model 16 heretofore described . two alternative experimental methods for realizing the calibration set are provided below . in the first method , spectral measurements of a target area of human skin are obtained using a nir reflectance instrument . biopsies of the scanned region are then obtained and examined histologically . the thickness and chemical composition of the key indicators specific to each tissue layer were included in the calibration set . using multivariate regression analysis techniques , a calibration model can then be developed to relate the spectral skin measurements , known as predictor variables , to the known skin layer thickness and chemical compositions , known as response variables . this technique uses a priori information regarding the general physiology of skin and exploits the inherent difference between skin layers and their compositions to develop a model that predicts skin layer thickness and composition noninvasively . the second approach is to develop a tissue model that adequately represents the fundamental absorbing and scattering characteristics of an in vivo tissue system . although , living tissue is a highly complex system , the transform from an in vivo system to a tissue model is made possible by an a priori knowledge of the primary absorbing and scattering species present in the living tissue system . because the model also includes a known thickness for each tissue layer , and since the concentrations of absorbing and scattering components are known , a monte carlo simulation may be used to simulate the photon propagation of light through the tissue model . the result of the monte carlo simulation is a diffuse reflectance measurement that is comparable to an actual reflectance measurement obtained experimentally . the tissue model must be validated in order to confirm that the model mirrors the complexity of the living tissue with sufficient accuracy to produce analogous results in application . a study was performed using ten subjects , five males and five females . nir absorbance spectra were collected using a custom spectrometer in diffuse reflectance mode . the pure component absorbance spectrum of water and fat were projected onto the measured spectrum in the 1100 - 1400 nm range and the resulting magnitudes are plotted , by sex , in fig2 . the figure shows a systematic difference in the relative magnitudes of the key indicators by sex . the subjects assorted into two distinct groups , with the males tending to exhibit high magnitudes of water absorbance , indicating a relatively thicker dermis , and low magnitudes of trigylceride absorbance , indicating a relatively thinner subcutaneous or adipose layer . conversely , the females tended to exhibit low magnitudes for water absorbance and high magnitudes for trigylceride absorbance , suggesting a relatively thinner dermis and a relatively thicker subcutaneous or adipose layer . such a systematic difference is consistent with that reported in the literature , le . a thicker layer of adipose tissue in females than in males and a thinner dermis in females than males [ see tan , et al ., op . cit .]. thus , the gross measurement of relative skin thickness through an nir diffuse reflectance measurement is amply demonstrated . quantification of the measurement is accomplished through calibrations based on prior in vivo measurements or monte carlo simulations as described above . the second method employs a general calibration model to predict the total skin thickness or the thickness of target layers on the basis of the measured absorbance spectrum . in overview , the method includes the following steps : measuring the nir spectrum of a target layer at a tissue site ; processing the nir spectral measurement through a general calibration model ; and as previously described , an estimate of total thickness is derived by summing the thickness estimates for the individual tissue layers . the general calibration model is based on a calibration set that includes spectral measurements , as previously described , made at a target tissue measurement site on a diverse group of individuals , and thickness measurements of the individual layers based on histological analysis of biopsy results or another commonly accepted method of skin thickness determination , pulsed ultrasound for example . the calibration model is developed using known methods , including principal component regression partial least squares regression and artificial neural network ( see h . martens , t . naes . multivariate calibration , new york : john wiley and sons , ( 1989 ); p . geladi , b . kowalski , partial least - squares regression : a tutorial , analytica chimica acta , vol . 185 , pp . 1 - 17 , ( 1986 )). new absorbance spectra are then processed through the calibration model to arrive at an estimate of skin thickness for the corresponding tissue sample . a study was performed involving nineteen volunteers of diverse age ( 21 - 55 years ) and sex ( sixteen males and three females ). skin fold thickness of each participant was measured on the forearm with research grade calipers of the type known as harpenden , manufactured by british indicators , ltd . nir scans of each subject were taken on the forearm and a calibration model for predicting the skin fold thickness was developed using partial least squares regression . the model was evaluated through cross - validation and the results are shown in fig3 . estimated versus actual skinfold thickness determination were plotted for each subject . the standard error of prediction was 1 . 42 , yielding a prediction accuracy of 70 percent . the results clearly demonstrate the feasibility of determining the thickness of a target layer from a general calibration model . the differences in skin thickness and the composition of the different layers produce a confounding effect in the noninvasive prediction of blood analytes . in one individual , at a particular time , an absorbance spectrum is representative of a distinct tissue volume that is sampled by the penetration of the light . when the target analyte for prediction is present in a particular layer it absorbs the light in a manner that is determined by its concentration and the pathlength of light within the particular layer . however , this pathlength is a function of the optical properties of the layer and the optical properties of the surrounding layers . therefore , knowledge of the thickness of individual skin layers and their optical properties can be used to reduce the interference resulting from this nonlinear variation . the skin thickness can be used in a classification system that develops calibrations specific to groups or classes of individuals based on tissue structure and state , fully described by malin , et al . in the previously cited related application , ser . no . 09 / 359 , 191 . however , in an alternative method for reducing interference due to non - linear variation , skin thickness and composition can be used with a nonlinear function to normalize the measured spectrum . the function can be determined from the light distributions in monte carlo simulations involving skin models of diverse composition and thickness . although the invention has been described herein with reference to certain preferred embodiments , one skilled in the art will readily appreciate that other applications may be substituted for those set forth herein without departing from the spirit and scope of the present invention . accordingly , the invention should only be limited by the claims included below .
a novel approach to measuring the overall and layer - by - layer thickness of in vivo skin tissue based on near infrared absorbance spectra is described . the different biological and chemical compounds present in the various layers of a tissue sample have differing absorbance spectra and scattering properties that enable them to be discerned and quantified , thus allowing an estimate of the thickness of the tissue being sampled . the method of the invention also yields the chemical composition of the absorbing and / or scattering species of each layer . additionally , a method of path length normalization for the purpose of noninvasive analyte prediction on the basis of skin thickness and layer constituents is provided .