Process and device for the determination of local dye concentrations and of scattering parameters in animal and human tissues

The invention relates specifically to a process for the determination of local dye concentrations in animal and human tissues. Light from two different wavelength regions (I, II), one of which (I) has a window for hemoglobin, is irradiated into a subregion of the tissue and the diffuse reflectance (MIO, MIIO) determined. Then, in another step, by means of the diffuse reflectance in the first wavelength region and at least one tissue-type-specific standard basic diffuse reflectance curve obtained in advance for both wavelength regions a tissue-person-specific standard basic diffuse reflectance curve (SIIO) for the second wavelength region (II) is determined, and by means of the determined tissue-person-specific standard basic diffusereflectance curve (SIIO) and the measured diffuse reflectance (MIIO) in the second wavelength region (II) a value for the hemoglobin concentration (KHb1) is obtained.

BACKGROUND AND SUMMARY OF THE INVENTION 
The invention relates to a process for the determination of local dye 
concentrations in animal and human tissues, in which light of differing 
wavelengths is irradiated into a subregion of the tissue, at least a part 
of the back-scattered light is collected, the diffuse reflectance 
(remission) is determined as a function of the wavelength and the 
concentration of dyes is determined from the spectral diffuse reflectance. 
Such a process is known, for example, from the dissertation "Bestimmung von 
Hamoglobin--Oxygenierung und relativer Hamoglobin--Konzentration in 
biologischen Systemen durch Auswertung von Remissionsspektren mit Hilfe 
der Kubelka-Munk-Theorie" ("Determination of hemoglobin oxygenation and 
relative hemoglobin concentration in biological systems by evaluation of 
diffuse reflectance spectra by means of the Kubelka-Munk theory") by 
Wolfgang Dummler, Erlangen, 1988. 
The term "local" concentration is to be understood here especially and 
exemplarily as the intracapillary region. 
The term "dyes" is to be understood as dyes intrinsic to tissues 
(pigments), especially hemoglobin, but also cytochromes and supplied dyes, 
for which the elution kinetics is then investigated. 
"Light of differing wavelengths" is generally the mixed light of a lamp 
(e.g., a xenon high-pressure lamp), but can also be, for example, the 
light of a tunable laser light source. The light is usually spectrally 
decomposed only after the diffuse reflection, and the intensity is 
evaluated as a function of the wavelength, the spectrally differing 
initial intensities being taken into consideration computationally. 
The term "subregion" is to be understood as a region with relatively small 
surface area, typically in the range of 50-100 .mu.m diameter. The depth 
extent in the tissue depends on numerous factors and is on the order of 
150 .mu.m (falloff to 1/e. As is described further below, however, the 
tissue volume from which the diffuse reflectance is obtained is both 
tissue-specific and equipment-specific and also depends on the hemoglobin 
concentration. 
As is described in detail in the cited dissertation by Dummler, the 
absolute measurement of the hemoglobin concentration, for example, is 
affected by considerable difficulties. Therefore, the invention creates a 
process and a device which makes it possible to determine substantially 
more exactly the dye concentration and other scattering factors in the 
tissue, especially the hemoglobin absolute concentration. 
This is achieved according to the invention in that, in one step, radiation 
from a first wavelength region in which the influence of the hemoglobin on 
the diffuse reflectance is small is irradiated and the diffuse reflectance 
in this wavelength region is determined, that in a separate step light 
from a second wavelength region in which the diffuse reflectance is 
dominated by the influence of the hemoglobin is irradiated into the same 
subregion of the tissue and the diffuse reflectance in this wavelength 
region is determined, that from the diffuse reflectance in the first 
wavelength region and at least one tissue-type-specific standard basic 
diffuse reflectance curve obtained in advance for both wavelength regions 
a tissue-person-specific standard basic diffuse reflectance curve for the 
second wavelength region is determined, and that from the determined 
tissue-person-specific standard basic diffuse reflectance curve and the 
measured diffuse reflectance in the second wavelength region a value for 
the hemoglobin concentration is obtained. 
The classification of the steps, e.g., as is to be found from the numbering 
in the claims, is done systematically. Digits after a colon are to signify 
alternatives of the step indicated in the first digit; following digits 
without a colon are substeps of a main step. The light measurements in the 
two wavelength regions I and II, 1.1. and 1.2., are systematically 
different (sub)steps but in practice occur simultaneously, the order being 
of no significance. The basic measurements (0. steps) generally occur 
before ("in advance of") the actual measurements, but in principle can 
also be performed subsequently, since the actual measurement values can 
also be stored. 
The term "basic diffuse reflectance" is understood here as the diffuse 
reflectance of the hemoglobin-free tissue, as can be found for example 
with a hemoglobin-free perfusion of the tissue. 
The term "tissue-type-specific" is understood as the special features which 
result from the special nature of the tissue (e.g., rat liver or human 
skin). The term "tissue-person-specific" designates values and curves in 
which the actual measurement of at least one of the two diffuse 
reflectance curves is already incorporated, even if that be only through 
the influence of the measurement on the choice from a family of curves 
determined in advance. 
The solution according to the invention has the specific advantage that, 
owing to the fact that in the first wavelength region where the hemoglobin 
has a window the basic diffuse reflectance is recognizable in 
comparatively unperturbed form, the influence of the basic diffuse 
reflectance in the second wavelength region where it is generally 
completely covered over by the hemoglobin influence can also be more 
exactly estimated and correspondingly eliminated. The more exact value 
thus obtained can be further refined in further process steps. 
Preferably, a family of tissue-type-specific standard basic diffuse 
reflectance curves is obtained in advance from tissue samples of the same 
tissue type, and the measured diffuse reflectance curve in the first 
wavelength region is assigned to the closest matching branch in the first 
wavelength region from the family of standard basic diffuse reflectance 
curves, and the associated branch of this standard basic diffuse 
reflectance curve in the second wavelength region is selected as the 
tissue-person-specific standard basic diffuse reflectance curve. 
Standard basic diffuse reflectance curves are understood as basic diffuse 
reflectance curves that were measured and stored "in advance" from a large 
number of tissues of the tissue type to be measured, e.g., by means of 
hemoglobin-free perfusion. In this embodiment of the invention, curves are 
determined which as a family of curves cover a large range of diffuse 
reflectances at one wavelength without the individual curves intersecting. 
From the family the curve is then selected which comes closest to the 
measured curve in the first wavelength region, and the other branch of 
this selected (tissue-type-specific) standard basic diffuse reflectance 
curve in the second wavelength region becomes through this selection the 
tissue-person-specific (standard) basic diffuse reflectance curve there. 
This has the special advantage that after such a family of curves is 
prepared it becomes possible in a simple manner to infer the (in itself 
unknown) behavior in the second wavelength region of the curve measured in 
the first wavelength region. 
In an especially preferred manner, the assignment of a standard basic 
diffuse reflectance curve from the family of standard basic diffuse 
reflectance curves obtained in advance in the first wavelength region to 
the measured diffuse reflectance curve is accomplished in that the 
standard basic diffuse reflectance curve with the value at a predetermined 
isosbestic wavelength in the first wavelength region which is equal to or 
closest to the measured diffuse reflectance value at that isosbestic 
wavelength is selected, and the value of the selected standard basic 
diffuse reflectance curve at a predetermined isosbestic wavelength in the 
second wavelength region is used as the value for the determination of the 
hemoglobin concentration. 
The values at the isosbestic wavelengths are taken because no additional 
error occurs there due to the (likewise still unknown) oxygenation of the 
hemoglobin. But these values also suffice for the stated purpose because 
the diffuse reflectance value from the diffuse reflectance curve at an 
isosbestic wavelength, corrected by the basic diffuse reflectance, already 
suffices to determine the concentration from the diffuse reflectance value 
using a suitably calibrated device. 
In an alternatively preferred manner, an averaged tissue-type-specific 
standard basic diffuse reflectance curve is obtained in advance from 
tissue samples of the same tissue type, and the measured diffuse 
reflectance curve in the first wavelength region is compared by ratio to 
the averaged standard basic diffuse reflectance curve, and, from the ratio 
obtained and the part of the tissue-type-specific averaged standard basic 
diffuse reflectance curve in the second wavelength region, a 
tissue-person-specific standard basic diffuse reflectance curve in the 
second wavelength region is obtained. 
Thus, in distinction to the previous alternative, now one typical curve is 
determined from a plurality of advance measurements of the basic diffuse 
reflectance of the tissue (e.g., by hemoglobin-free perfusion) over both 
wavelength regions, which for that reason is also designated as the 
"averaged" standard basic diffuse reflectance curve (although the curves 
of the family of curves may in turn also have resulted from averagings). 
"Comparison by ratio" is to be understood in any case not only as the 
formation of a mathematical ratio, but rather numerous methods are 
conceivable by which from the deviations of the behavior of the measured 
diffuse reflectance curve in the first wavelength region from the behavior 
of the averaged standard diffuse reflectance curve in the first wavelength 
region via the behavior of the averaged standard diffuse reflectance curve 
in the second wavelength region it is possible to infer the imaginary 
continuation of the measured curve (considered as the basic diffuse 
reflectance curve in zeroth approximation) as the tissue-person-specific 
basic diffuse reflectance curve in the second wavelength region. 
In an especially preferred manner, the comparison by ratio of the averaged 
standard basic diffuse reflectance curve in the first wavelength region to 
the measured diffuse reflectance curve (in the first wavelength region) is 
accomplished in that the value of the averaged standard basic diffuse 
reflectance curve in the first wavelength region at a predetermined 
isosbestic wavelength in the first wavelength region is compared by ratio 
to the measured value of diffuse reflectance at that isosbestic 
wavelength, and by means of the obtained ratio the value of the averaged 
standard basic diffuse reflectance curve at a predetermined isosbestic 
wavelength in the second wavelength region is used to obtain a value of 
diffuse reflectance at that isosbestic wavelength, which is used as the 
value for the determination of the hemoglobin concentration. 
The advantage of the use of the values at isosbestic wavelengths was 
already explained. The diffuse reflectance value, which allows the 
hemoglobin concentration to be inferred, is generally determined by 
subtracting the value obtained at the isosbestic wavelength in the second 
wavelength region from the measured value of diffuse reflectance at that 
wavelength. 
In a preferred manner, in a continuation of the process, the measured curve 
in the first wavelength region is corrected by means of the value obtained 
for the hemoglobin concentration, whereby a second, improved approximation 
is obtained for the tissue-person-specific basic diffuse reflectance in 
the first wavelength region. 
In the above process, the measured diffuse reflectance curve in the first 
wavelength region, which still contained the influence (which of course is 
small there) of the hemoglobin concentration, was a "zeroth approximation" 
(or zeroth order) of a tissue-person-specific basic diffuse reflectance 
curve in the first wavelength region. This zeroth approximation can now be 
improved by eliminating the hemoglobin concentration (which in turn is 
known in first approximation from the above process steps) from the curve. 
The further approximation thus obtained is advantageously incorporated 
into the above-described process steps in place of the measured diffuse 
reflectance curve. 
Thus, in an especially preferred manner, steps 2 to 4 are performed with 
the improved curve instead of the measured diffuse reflectance curve, 
whereby a better approximation value is obtained for the hemoglobin 
concentration and a further improved curve is obtained as 
tissue-person-specific basic diffuse reflectance in the first wavelength 
region. In a preferred manner, the above steps 2. to 4. are repeated n, 
where n is a predetermined number, times, using the improved values and 
curves as a basis in each instance. 
The region from 630 nm to 1000 nm is preferred as a wider first wavelength 
region. 
The region from 750 nm to 850 nm is preferred as a narrower first 
wavelength region. Hemoglobin has a window in these regions, i.e., its 
influence on the diffuse reflectance is small. 
The region from 500 nm to 620 nm is preferred as a wider second wavelength 
region. 
The region from 550 nm to 570 nm is preferred as a narrower second 
wavelength region. In the latter two regions, the influence of hemoglobin 
on the basic diffuse reflectance is large. 
The invention also relates to a process for the determination of the 
oxygenation of hemoglobin, especially using one or more of the curves from 
one or more of the above processes. The determination of the oxygenation 
of hemoglobin with high accuracy is also of special significance in the 
monitoring of life processes by means of spectrophotometry. 
In this connection, according to the invention, a "pure" hemoglobin curve 
is obtained from a tissue-person-specific standard basic diffuse 
reflectance curve and the measured diffuse reflectance curve in the second 
wavelength region; a family of "pure" hemoglobin curves having been 
obtained in advance in the range of from 0% to 100% oxygenation by 
superposition of two pure standard hemoglobin curves, namely for 0% and 
100% oxygenation, with different weightings; the "pure" hemoglobin curve, 
after normalization to 1, is compared to the likewise normalized standard 
hemoglobin curves of the family, the closest matching one is picked out, 
and its oxygenation is assumed as the value of the oxygenation for the 
measured curve. 
In an alternatively preferred process for the determination of the 
oxygenation of hemoglobin, especially using one of the concentration 
values and especially one of the curves from one of the previously 
described processes, a two-dimensional family of comparison curves is 
prepared in advance by means of a plurality of measurements on the same 
tissue type with hemoglobin of differing concentrations and differing 
oxygenations, the comparison curves with hemoglobin concentrations in the 
vicinity of the determined concentration are searched throughout the 
entire range of oxygenation, and the best matching of the comparison 
curves yields an assumed value for the oxygenation and an improved value 
for the concentration. 
In this manner, reliable values for important parameters can in turn be 
obtained simply and quickly with the aid of the measured diffuse 
reflectance and standard values known in advance. 
In a preferred manner, the values for the concentration and the oxygenation 
obtained in the manner just described are used in the described step for 
obtaining an improved tissue-person-specific basic diffuse reflectance 
curve. 
In an especially preferred manner, the measured diffuse reflectance curve 
from the second wavelength region is normalized to the closest matching 
curve from the above-mentioned two-dimensional family used to determine 
the oxygenation, the difference between the two curves is plotted versus 
wavelength and used as a measure of the distortion to determine the 
penetration depth of the irradiated light, i.e., of the volume V covered 
by the light. 
It has turned out that this distortion can be used as a measure of the 
penetration depth. Corresponding distortion curves determined in advance 
are stored, the corresponding volume is assigned to them, and then the 
distortion curve obtained by the comparison is assigned to the closest 
matching curve from the stored distortion curves and thus the volume is 
determined. 
In an especially preferred manner, the Erlanger light-guide 
microspectrophotometer (Erlanger Mikrolichtleiterspektrophotometer 
(EMPHO)) is used for the above measurements. It is described in more 
detail further below. The graded-density interference filter disk used in 
it covers preferably both the first wavelength region and also the second 
wavelength region. In that way, the measured diffuse reflectance curves of 
the first and second wavelenqth regions can be obtained in one rotation of 
the disk. 
In a preferred manner, the light-guide microspectrophotometer has means for 
absolute calibration of the illuminating and detecting system. The 
absolute calibration is especially important because the measurements made 
in advance, which should be made with the same device or must be converted 
in a device-specific manner, must occur under defined conditions 
comparable to those of the actual measurement. In particular, such means 
are a white standard and a standard light source, which are explained 
below. 
The invention relates also to a device for the determination of size 
variations of tissue particles. The observation of such variations, e.g., 
of the size change of mitochondria, is of special practical importance 
because it makes it possible, for example, to detect a cerebral edema at 
an early stage. This is accomplished by means of a device with a light 
guide radiating light into the tissue, at least two light guides at 
different radial distances from it [the first light guide] which receive 
the back-scattered light and which preferably are disposed along a line on 
both sides of the illuminating light guide, and an evaluation unit for 
each of the light guides which determines and evaluates the time variation 
of the back-scattered intensity relative to the other light guides. 
Such an evaluation unit can be built analogously to the Erlanger 
light-guide microspectrophotometer. A flattening or other deformation of 
the back-scattering characteristic can then be recognized, which in turn 
allows one to infer the variation of the particle size.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
FIG. 1 shows on the right a first measured curve MIO (M is to indicate 
"measured", I is to indicate the wavelength region I (here 750-850 nm), 
and the digit 0 indicates that it involves a curve which can be considered 
as the zeroth approximation of the basic diffuse reflectance curve). In 
I.sub.o /I is used here as a measure of the diffuse reflectance. The curve 
was measured on rat livers with the Erlanger light-guide 
microspectrophotometer, which is explained in more detail in, among other 
places, the dissertation "Optische Streuung an biologischen Partikeln und 
Zellen" ("Optical scattering from biological particles and cells"), 
Erlangen 1985, by one of the inventors, Frank, but also hereinbelow. 
The curve exhibits only a slight influence of the hemoglobin, since 
hemoglobin has a window in region I. 
The term "basic diffuse reflectance" is understood here as the diffuse 
reflectance of the hemoglobin-free tissue, as can be found, for example, 
for a hemoglobin-free perfusion of the tissue. This basic diffuse 
reflectance is still tissue-type-specific, depends on the redox state of 
the remaining cell pigments and possibly added dyes, and is also still 
tissue-person-specific, although only to a slight extent. Accordingly, it 
is necessary to determine the actual "true" basic diffuse reflectance as 
exactly as possible in order to be able to determine the concentration, 
oxygenation and redox state of different pigments and dyes. 
The measured curve MIO (systematically considered in a process step 1.1) 
represents a (zeroth) approximation order in the process, because it is 
still dependent on the influence (which in the selected wavelength region 
I from 750 to 850 nm is of course small) of the hemoglobin concentration 
(KHb) and of the oxygenation (HbO.sub.2 /Hb). 
In another step (2.), a corresponding standard basic diffuse reflectance 
curve in the wavelength region II is selected for the measured basic 
diffuse reflectance curve MIO. 
For the determination of the so-called "standard" basic diffuse reflectance 
curves (so to say, in a 0th process step), a large number (about 100) of 
in-vivo and in-vitro samples which have undergone hemoglobin-free 
perfusion are used to determine which diffuse reflectance curves result in 
region II for particular diffuse reflectances in region I. Thus, a large 
number of families of values is determined, and the families of values 
contain values of a curve for the different wavelengths from wavelength 
regions I and II. 
In the actual table (FIG. 4), a sequence at an isosbestic point of the 
hemoglobin in region II is uniquely assigned to a sequence (one value each 
from the families of values forming a curve) of diffuse reflectances at an 
(here "the") isosbestic wavelength of the hemoglobin in wavelength region 
I (in such a way that the values of the value pair lie on the same 
standard basic diffuse reflectance curve). Thus, the table represents a 
narrowest section from the associated curves at the indicated wavelengths. 
The isosbestic wavelength of the hemoglobin (815 nm) was selected in 
wavelength region I, since the influence of the oxygenation of the 
hemoglobin on the diffuse reflectance in wavelength region I also is 
greater than the influence of the oxidation of the cytochromes, which 
latter influence changes appreciably only at an oxygen partial pressure of 
less than 5 torr. The choice of the isosbestic wavelength results in 
independence from the (in this step still unknown) oxygenation of the 
hemoglobin. The error of the zeroth approximation, the measured curve MIO, 
depends there only on the concentration of the hemoglobin. 
Thus, for the assignment of a standard basic diffuse reflectance curve 
(SIIO) of zeroth approximation in wavelength region II, the value of the 
sequence at the isosbestic wavelength in wavelength region I is selected 
which is equal to or closest to the measured value at the isosbestic 
wavelength on the curve MIO, and by means of Table 2 there is assigned to 
the latter the associated value of the sequence in wavelength region II 
and thus also the entire branch of the corresponding standard basic 
diffuse reflectance curve in II. 
Alternatively, in a process step 0.2 a single "averaged standard basic 
diffuse reflectance curve" averaged from all measured standard basic 
diffuse reflectances is formed, and, for the differences (or factors) in 
comparison to other diffuse reflectances at the isosbestic wavelength in 
region I, tables are produced (using a large number of standard basic 
diffuse reflectance curves covering both wavelength regions) which assign 
to a difference (or a factor) a difference (or a factor) at wavelengths in 
the 2nd wavelength region, especially again at an isosbestic wavelength 
therein. Then, in the second process step, alternatively, the (step 2.2) 
the difference (or the ratio) between the diffuse reflectance of the 
measured curve MIO and of the averaged standard basic diffuse reflectance 
curve at the isosbestic wavelength is found, and the averaged standard 
basic diffuse reflectance curve in the second wavelength region at the 
isosbestic wavelength is loaded with the associated factor or summand on 
the basis of the table so as to obtain a curve, or at least its value at 
an isosbestic wavelength in wavelength region II, which represents the 0th 
approximation of the (standard) basic diffuse reflectance in the second 
wavelength region II, SIIO. 
An isosbestic wavelength is also selected in region II (specifically 586 nm 
in the exemplary embodiment) so as to be independent of the unknown 
oxygenation. 
FIG. 1 shows in the left half a measured diffuse reflectance curve at the 
same tissue location (systematically considered in a step 1.2.), obtained 
in the same passage of an interference graded-density filter disk (see 
below, FIG. 13). It is designated as MIIO as the measured curve in 
wavelength region II and zeroth approximation. Also drawn to the left in 
FIG. 1 is the standard basic diffuse reflectance curve SIIO belonging to 
the measurement value determined in the first wavelength region I, which 
also as described above is present as a table (a section of which is shown 
in FIG. 2) or is determined from the comparison of the averaged standard 
basic diffuse reflectance curve in region I with the curve MIO, as 
alternatively described above. At the isosbestic wavelength, from the 
measured total diffuse reflectance (curve MIIO), which is composed of the 
basic diffuse reflectance (represented in zeroth approximation by the 
standard basic diffuse reflectance) and the hemoglob-independent diffuse 
reflectance, by subtraction of the value of the standard basic diffuse 
reflectance curve SIIO at the actual isosbestic wavelength (586 nm) from 
the value of the measured diffuse reflectance curve MIIO, the hemoglobin 
diffuse reflectance and thus a measure for the hemoglobin concentration in 
first approximation, KHb1, is obtained (in step 3.). Thus, a first 
approximation value, KHb1, for the hemoglobin concentration is obtained. 
This concentration value KHb1 is used to correct the measured curve in 
region I, MIO, and one obtains for the basic diffuse reflectance a 
corresponding better curve of first approximation, GI1, by subtracting 
from MIO (in a step 4.) the (additional) amplitude value produced by the 
hemoglobin concentration at the isosbestic wavelength of 815 nm at that 
concentration. The resulting value is used with the table (FIG. 4) (or the 
alternative process step 2.2) to select a better-matching standard basic 
diffuse reflectance curve SII1 (see FIG. 1, left), which in turn serves to 
improve the value of the hemoglobin concentration to a value KHb2. The 
latter is used in turn, in the described manner, to determine an improved 
basic diffuse reflectance curve in region I, GI2. By multiple repetition 
one can finally obtain a highly improved value for the hemoglobin 
concentration, KHbn. In another step the oxygenation is determined, 
possibly with simultaneous improvement of the value for the concentration. 
First of all, the standard basic diffuse reflectance curve corresponding to 
the last iteration stage is subtracted from the measured curve MIIO to 
obtain the "pure" hemoglobin curve, HIIO. 
By superposition of two pure standard hemoglobin curves, namely for 0% and 
for 100% oxygenation, with different weighting, a family of "pure" 
hemoglobin curves is obtained in the range of 0-100% oxygenation. 
Then, the corrected measurement curve, HIIO, after normalization to 1, is 
compared to the likewise normalized curves of this family and the closest 
matching one is selected (e.g., by the least-squares method). 
The degree of oxygenation of the selected curve is assumed as the degree of 
oxygenation of the measured curve MIIO. 
Alternatively, before beginning the actual measurement, standard diffuse 
reflectance curves were determined with the same device with which the 
actual measurement is performed, using a very large number of measurements 
on the same tissue type, which was perfused with hemoglobin of differing 
concentrations (from 0 to 20%) and differing oxygenations (0 to 100%). The 
standard diffuse reflectance curves, which in turn are averaged from a 
large number of measurements for a particular concentration and 
oxygenation, are arranged in a table or matrix so that, for example, the 
rows contain the different concentrations at the same oxygenation and the 
columns contain the different oxygenations at the same concentration. This 
is illustrated schematically in FIG. 5. 
The measured curve MIIO is now compared with the curves in the table. In an 
exemplary and preferred manner, the column corresponding to the first 
approximation of the hemoglobin concentration and one each or in the 
exemplary embodiment two each of the adjacent columns are searched for all 
values of the oxygenation. 
For the comparison of the curves, in one embodiment of the process the 
integral of the area under the standard diffuse reflectance curves can be 
compared with the integral under the measured curve MIIO. 
Alternatively, the curves are compared by the least-squares method. 
The field of the matrix with the optimally matching curve yields a 
hemoglobin concentration value, second approximation, KHb2, and a 
hemoglobin degree of oxygenation, first approximation, KHbO.sub.2 1. 
These values are now, for example, used again to get improved values for 
the basic diffuse reflectance curve and the concentration. 
Here also it is evident that the process can be continued until a 
convergence which is sufficient with respect to the realistic measurement 
accuracy is reached. 
The hemoglobin oxygenation simultaneously allows one to infer the oxidation 
of the cytochromes. Furthermore, the basic diffuse reflectance curve after 
subtraction of the hemoglobin influence allows a more exact determination 
of other parameters in the tissue. 
At very low oxygenation, i.e., oxygen partial pressures &lt;5 torr measured in 
the tissue, other basic diffuse reflectance curves must be used as a 
basis, but this does not change anything in the basic process. 
FIG. 2 shows, at magnified scale, measured curves from wavelength region I 
corresponding to the curve MIO, one for high hemoglobin oxygenation 
(M'IO.sub.h) and one for low hemoglobin oxygenation (M'IO.sub.n). 
FIG. 3 shows, at magnified scale, standard basic diffuse reflectance curves 
corresponding to the curves SII, one at high (SII.sub.h) and one at low 
(SII.sub.n) oxidation of the respiratory enzymes. 
More-exact statements about a "concentration" can only be made by taking 
into consideration of the tissue (micro)volume covered by the measurement. 
The covered volume depends on the following parameters: 
1. the utilized wavelength and, very generally, the characteristic of the 
utilized light source (luminous field density, intensity, stability) 
2. the transmission characteristics of the light guide radiating into the 
tissue (acceptance angle .alpha., length L, diameter d, material) 
3. the scattering characteristic of the tissue and the absorption behavior 
of the dye or dyes 
4. the transmission characteristics of the (detecting) light guide 
receiving the back-scattered light (acceptance angle .alpha., length L, 
diameter d, material) and of the following detection system 
5. the sensitivity of the light-measuring system and/or of the 
photomultiplier. 
FIG. 6 shows schematically the illuminating lamp 2, the illuminating light 
guide 4, the detecting light guide 6 and the photomultiplier 8. The volume 
into which the illuminating light guide 4 radiates in the tissue is 
indicated by Eh for a high Hb concentration and by En for a low one; the 
volume from which the detecting light guide 6 can receive light, taking 
into consideration the sensitivity of the photomultiplier 8, is designated 
by R.sub.h and R.sub.n, respectively. The intersection volume, V.sub.h and 
V.sub.n respectively, is the volume on which the concentration measurement 
is based. A quasi-diffuse illumination is achieved by high luminous 
density. 
It is assumed that the device used to produce the utilized tables 
(matrices) is the same as that used for the actual measurement, so that to 
that extent there is no change in the volume or only factors that can be 
factored out are changed. 
Information about the volume covered under certain conditions can also be 
presented in tabular form. The corresponding values can be obtained by 
means of measurements on sections of the tissue or in scattering chambers 
with simulated tissue. 
The above-mentioned concentration values are based on volumes which are 
obtained from the tables as empirical values. However, the volumes can 
also be corrected by means of a separate procedure. 
According to the invention, the distortion of the measured hemoglobin curve 
MIIO compared to the standard diffuse reflectance curve RSII1 determined 
from the matrix (table) 2 (see FIG. 5) is used as a measure for the 
penetration depth and thus for the covered volume. For that purpose, the 
measured hemoglobin curve MIIO is normalized to the standard curve 
determined at the isosbestic wavelength and the difference is plotted 
versus wavelength. 
FIG. 7 shows (schematically) an example of a possible standard diffuse 
reflectance curve RSII1 with two examples for possible measurement curves 
MIIO, and FIG. 8 shows the difference or distortion curves resulting from 
each of them. 
In corresponding advance measurements with the same device a matrix of such 
distortion curves was prepared and each field of the matrix was assigned 
to a covered volume V (see FIG. 6). That matrix is not shown here. 
In this manner the volume can be determined and in turn used as a 
correction factor for the previously determined values of the 
concentration and oxygenation. 
With a light-guide arrangement shown in FIG. 9 and its use in a special 
process, the invention also makes it possible to determine the variation 
of particle sizes in the tissue. This determination is of special 
practical significance. For example, it can be used to ascertain a size 
variation of the mitochondria. A preferred embodiment of the device is 
shown schematically in FIG. 9. 
The device consists of an arrangement of a centrally disposed illuminating 
light guide 20 with a diameter of about 250 .mu.m and detecting light 
guides, about 70 .mu.m in diameter in the example, disposed along a line. 
With it one can determine the distribution of the back-scattered light in 
a cross-section of the back-scattering volume, possibly after factoring 
out the angle distortion resulting from the given arrangement. However, 
since the distribution and intensity of the light in this back-scattering 
volume varies distinctly with the particle size, a comparison of the 
diffuse reflectance values obtained from the different detecting light 
guides 21 to 30 in the course of time enables one to infer the variation 
of the particle size. 
With the assumption of radially symmetric conditions it is also preferred, 
rather than providing one or two light guides, each at a particular 
distance (and thus (cone) angle), instead to provide a circle of light 
guides with a radius equal to the distance. In that way, it is possible to 
determine the received light power associated with a particular distance, 
which can be made feasible by connecting together the light guides lying 
on a circle and evaluating them together. 
FIG. 10 shows the large variation of the intensity of the back-scattered 
(180.degree.) light as a function of the particle size (0.1-2 .mu.m). 
Detection of this variation by monitoring the time variation, especially 
the relative [variation] at the different receiving light guides, thus 
makes possible a reliable monitoring of the variation, e.g., of the size 
of the mitochondria and thus, e.g., a timely warning of the development of 
cerebral edemas. 
FIG. 11 shows graphically the change in the distribution of the light, 
which likewise can be used for the evaluation of the relative diffuse 
reflectances on the individual or pairs or circles of receiving light 
guides (here: 21 to 30). 
In another, especially preferred embodiment, a central illuminating light 
guide 20 and a field of, for example, 10.times.10 receiving light guides 
is provided (cf. also FIG. 12). This makes possible the measurement of 
entire topographies of oxygenation and dye distributions. 
The items of information coming from the individual light guides are first 
evaluated individually, as described above in detail for one receiving 
light guide, and the results then yield a topogram of the Hb 
concentration, a topogram of the HbO.sub.2 concentration and basic diffuse 
reflectance topograms. 
The interrogations of the light guides can be simultaneous, which then 
makes necessary a corresponding number of the evaluation units described 
below. However, given the brief period in which a complete spectrum is 
recorded in both wavelength regions (about 1/100 s), the light guides can 
also be interrogated in succession, which results in a time difference of 
ca. one second, which frequently is acceptable. 
The time variation in connection with the angle-dependent corrected spatial 
diffuse reflectance graph obtained by the arrangement again enables one to 
reach conclusions about the change of the particle size. 
Evaluation of the angle dependence, as concerns both the distance from the 
illuminating light guide and the circumference of circles around the 
illuminating light guide, can be used to determine spatial asymmetries. 
In another embodiment, which is illustrated concretely in FIG. 12, 
illuminating light guides 32-38 are also located in the middle of the 
lateral edges, whereby further information about the scattering behavior 
in the tissue can be obtained. The volume irradiated by the central light 
guide and the volumes detectable by each of the detecting light guides are 
illustrated for specific examples in the drawing. 
FIG. 13 shows the basic design of the Erlanger light-guide 
microspectrophotometer. The light of a xenon high-pressure lamp 40 (e.g., 
XBO 75 W/2, Osram), which is powered by a supply system 42 (stabilized 
power supply), is irradiated via an optical system 44 into the 
illuminating light guide 4. The latter is combined with the detecting 
light guide 6 in such a way that the respective end faces lie in one plane 
and immediately next to one another (not shown in FIG. 13). The pair of 
light guides (or the arrangement from FIG. 9 or FIG. 12) is then placed on 
the tissue surface 46. Via the receiving light guide 6 the light reaches 
an interference graded-density filter disk 48. In the invention, the 
latter encompasses the wavelength range of from 500 up to 850 nm, in 
contrast to the interference graded-density filter disks used heretofore 
for tissue spectrophotometry. It is obvious that then the measurements of 
the curves MIO and MIIO can occur practically simultaneously and the order 
depends only on the rotation direction of the filter disk. The light of 
the wavelength region just let through by the interference graded-density 
filter disk 48 (the resolution is about 2 nm) is sent via a light guide 50 
to a photomultiplier 52. Via an amplifier system 54 the signal arrives at 
an analog-to-digital converter 56 and, after digitization, it is sent to a 
computer (also in 56) for further processing. 
As to which wavelength is involved in each instance, that is determined by 
disposing on the shaft of the motor 58 driving the filter disk 48 a 
decoder disk 60 and sending the control signals to an EPROM 62. The EPROM 
62 converts the control signals into trigger signals for the digitization 
of the measurement signal by the A/D converter 56. The decoder disk also 
produces a pulse which marks the beginning of each disk rotation and which 
initializes the digitization by the A/D converter. 
The present process works with absolute values of the diffuse reflectance. 
Therefore, special attention is paid to the calibration of the entire 
arrangement. 
FIG. 14 shows the device for establishing a white spectrum. The spectral 
distribution of the light of the xenon arc lamp, the transmission 
characteristics of the optical elements (lenses, light guides) and the 
spectral sensitivity of the photomultiplier yield a wavelength-dependent 
response function for white light. This can be measured by means of the 
spectrum of a white standard, here BaSO.sub.4, using the known device 
shown in FIG. 14. 
In the arrangement used for the measurement, the illuminating (4) and 
receiving (6) light guides are placed vertically in a drop of immersion 
fluid 70 (0.9% NaCl) on a glass plate 72. The glass plate provides a fixed 
distance to the white standard 74. The intersection region 76 of the light 
cones corresponds to the volume V. To establish the intensity range, a 
wavelength-dependent dark curve must be stored. 
The correction of the spectrophotometric measurements is performed in four 
steps. For this, reference is made to FIG. 15 and 16. 
1. The recorded measured spectrum RS is subtracted from the dark curve DC. 
2. The spectrum of the BaSO.sub.4 white standard (BaSt) is also subtracted 
from the dark curve (DC) (DC-BaSt=TF). 
3. The division (DC-RS)/(DC-BaSt) yields the corrected spectrum CS (FIG. 
16). 
4. For the display the corrected spectrum is multiplied by -1. 
In order to obtain absolute values of the concentration and because the 
measurements of the curves MIO and MIIO, on the one hand, and the 
measurement of the standard basic diffuse reflectance curves and other of 
the described comparison curves, on the other hand, may be performed at 
completely different times, an absolute calibration of, for example, the 
photomultiplier and other light-measuring devices that may be used is 
especially important. Used for that purpose is a standard light source, 
preferably in the form of a beta light, in which zinc sulfide or some 
other radioluminescing substance is excited by radioactive decay products, 
particularly beta rays of tritium. This is described in more detail in the 
older German patent application "Normallichtquelle" ("Standard light 
source") of the same applicant and the same inventors, official file 
number P 38 16 489.2 33, to which express reference is made.