 |
Description  |
|
|
BACKGROUND OF THE INVENTION
A reliable, quantitative and non-invasive method for the characterization
of the molecular changes associated with early cataractogenesis in-vivo
has long been an important goal of human clinical cataract research. Such
a method would allow researchers and physicians to: (a) assess the
effectiveness of putative anticataract reagents; (b) evaluate the
cataractogenic role of pharmacologic agents or radiation used in the
treatment of systematic disease; (c) characterize early cataract in
epidemiologic studies of human or animal populations subject to
differential cataractogenic stress; and, d) provide a quantitative basis
for the medical decision to intervene surgically or pharmaceutically in
the treatment of cataract.
In 1975, T. Tanaka and G. Benedek, ("Observation of Protein Diffusivity in
Intact Human and Bovine Lenses with Application to Cataract," Invest.
Ophthal. 14, 449-456 (1975).) showed that the Brownian motion of proteins
in excised human and bovine lenses could be measured optically using the
method of quasielastic light scattering spectroscopy. Following this work,
T. Tanaka and C. Ishimoto, ("In Vivo Observation of Protein Diffusivity in
Rabbit Lenses," Invest. Opthal. and Vis. Sci. 16, 135-140 (1977).)
demonstrated in 1977 that it was possible to conduct quasielastic light
scattering measurements safely in the lens of the eye of a living rabbit.
In 1984, Nishio et al., ("In Vivo Observation of Lens Protein Diffusivity
in Normal and X-Irradiated Rabbit Lenses," Exp. Eye Res. 39, 61-68
(1984).) demonstrated that quasielastic light scattering could be used in
vivo in the rabbit to detect changes in mean protein diffusivity as a
function of position and age in the lens. Further observations showed that
the cataractogenic insult of X-irradiation upon the rabbit lens produced
dramatic changes in the form of the autocorrelation function of the
scattered light at a very early stage in the cataractogenic process. This
change in the autocorrelation function, the mathematical heart of
quasielastic light scattering analysis, demonstrated that the
X-irradiation was responsible for drastic changes in the diffusivity of
the protein scattering elements undergoing Brownian movement within the
ocular tissue. Both Nishio and the 1977 Tanaka team observed that these
altered correlation functions had a form different from that expected for
the Brownian motions of a single type scatterer. However, neither
undertook a quantitative analysis of the information contained in the
non-exponential character of the autocorrelation function observed.
In 1986, T. Libondi et al., ("In Vivo Measurement of the Aging Rabbit Lens
Using Quasielastic Light Gathering," Curr. Eye Res., Vol. 5, 6, 411-419
(1986).) showed that the form of the autocorrelation function of the
scattered light from a living rabbit eye indicated the presence of at
least two distinct diffusing species within the rabbit lens. One species'
had a diffusivity corresponding to the .alpha.-crystalline protein. The
other was a much more slowly diffusing species of the type discovered
in-vitro by M. Delaye et al., ("Identification of the Scattering Elements
Responsible for Lens Opacification in Cold Cataracts," Biophys. J. 37,
647-656 (1982).) in 1982.
A recently discovered method of cataract detection comprises irradiating a
measurement location of a lens with a laser, collecting light scattered
from the lens at the measurement location, analyzing the collected light
using an autocorrelator or spectrum analyzer to determine the relative
amount of light scattered from different protein species in the lens, and
analyzing this data to determine the degree of cataract formation at the
measurement location in the lens. A more detailed description of the
method is given in copending Application Ser. No. 07/091,658; filed Sept.
1, 1987 now abandoned.
SUMMARY OF THE INVENTION
The present invention provides a method for non-invasive in-vivo inspection
of ocular tissue. More specifically, a laser is used to provide a low
power, coherent and uniform beam of light which is guided to a measurement
location in the eye of a subject for scattering by ocular protein
molecules. The scattered light is viewed and a portion of it is collected.
The intensity of the light scattered by the measurement location is
determined and this value is compared to a value representative of the
intensity of light scattered by a normal, clear, lens region. By
determining the intensity of light scattered from the measurement location
and comparing it to a standard value, it is possible to accurately and
quantitatively estimate the degree of cataractogenesis occurring in that
location of ocular tissue.
Thus, the present invention pertains to a method for in-vivo inspection of
ocular tissue comprising:
(a) providing a source of substantially monochromatic, coherent, collimated
light;
(b) focusing light from the source on a first measurement location of a
subject's ocular tissue;
(c) determining the intensity of light scattered by the ocular tissue at
the measurement location (I.sub.tot);
(d) comparing the value of I.sub.tot to a value representative of the
intensity of light scattered by a normal clear lens (I.sub.f.sup.o); and,
(e) using the numerical value of the ratio I.sub.tot /I.sub.f.sup.o to
determine the degree of cataract development at the specific site.
The method for in-vivo inspection of ocular tissue described herein has a
number of advantages over currently known techniques for determining the
degree of cataractogenesis. Unlike the present method, visual observation
of the eye lens using a slit lamp microscope is not a quantitative method,
nor is it sensitive enough to detect the earliest changes in scattered
light produced by the first stages of cataract formation. Current
photographic methods are useful for characterizing later stages of
cataractogenesis. However, the instrumentation is expensive and
insensitive to early cataractogenic changes due to the logarithmic
response of photographic film.
The above and other features of the invention including various novel
details of combination of parts will now be more particularly described
and pointed out in the claims. It will be understood that the particular
cataractogenesis detection method embodying the invention is shown by way
of illustration only and not as a limitation of the invention. The
principles and features of this invention may be employed in various and
numerous embodiments without departing from the scope of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic representation of an optical scattering analyzer for
the study of the temporal fluctuations of laser light scattered from the
lens in vivo.
FIG. 2 is a plot of I.sub.f * versus age at the position of the anterior
nucleus in 29 subjects.
FIG. 3 is a plot of I.sub.tot versus age at the position of the central
nucleus in 32 subjects.
FIG. 4 is a plot of I.sub.tot versus I.sub.s * for five preoperative
cataract patients.
FIG. 5 is a plot of I.sub.tot versus I.sub.s at the position of the central
nucleus in 28 subjects.
DETAILED DESCRIPTION OF THE INVENTION
The proteins present in ocular tissue undergo random diffusive movement due
to continuous collisions with nearby molecules. When a coherent, steady
beam of light is scattered by the moving proteins, the intensity of the
scattered light fluctuates in time. The movements of the proteins
determine the rate of intensity fluctuation. As it has been determined
that small proteins generally diffuse faster than larger proteins or
protein aggregates, it is possible to examine the detector current
fluctuations to determine the relative intensity of light scattered from
the small protein species and the large protein species within the lens
tissue, and to determine as well the relative amounts of these two species
and their individual diffusivities.
The aggregation of small proteins within the lens is the very first stage
in the process of cataractogenesis. By using information obtained from the
light scattered by the various fast and slow moving protein species, it is
now possible to interpret, in a clear and unambiguous manner, the meaning
of the components contained in the autocorrelation function or the power
spectrum. This information may be used to generate an algorithm which
provides a universal curve for cataract development. By locating the
position on this curve corresponding to each selected position in the
lens, it is possible to characterize in a very simple manner the precise
degree of cataractogenesis occurring at that selected point in the lens.
In effect, this invention permits the decoding of the information
contained in the random intensity fluctuations in the light scattered from
the lens. When placed on the disclosed universal curve, the decoded
information has been shown clinically to provide an accurate quantitative
measure of cataract development on a molecular level long before it could
be detected visually by either the subject or the physician.
Brownian motion is defined as the motion of macromolecules caused by
thermal agitation and the random striking by neighboring molecules in a
solution. In the lens of the human eye, protein molecules undergoing
Brownian motion may be recorded and analyzed by quasielastic light
scattering.
In quasielastic light scattering (QLS), the temporal fluctuations in
intensity of light scattered by a selected small volume in the lens which
is illuminated by an incident laser beam are studied. The scattered light
intensity fluctuates in time because of the Brownian motion of the
scattering elements. In the case in which the laser beam illuminates the
lens of the eye, the scattering elements are the molecular constituents of
the fiber cells within the lens. These constituents are principally
globular proteins called crystallins.
The light intensity fluctuations are detected by collecting the light
scattered from a well-defined, illuminated volume in the eye lens and
focussing this light onto the surface of an optical square law detector
such as a photomultiplier tube or solid-state photodiode. The output of
the detector is a photo-electric current whose temporal fluctuations are
in synchrony with the fluctuations in the scattered light intensity. The
temporal fluctuation in the photocurrent can be analyzed mathematically by
obtaining the autocorrelation function or the power spectrum of the
photocurrent. From the mathematical form of the autocorrelation function
or the power spectrum of the photocurrent, it is possible to determine the
diffusivity of the scattering elements undergoing Brownian movement.
The autocorrelation function may be determined by using an autocorrelator
to analyze the fluctuations in the intensity of the laser light scattered
by the ocular tissue Correspondingly, the power spectrum may be determined
using a spectrum analyzer. The random motions of the crystalline proteins
within the lens give rise to concentration fluctuations, which in turn
give rise to fluctuations in the intensity of the scattered light. This
scattered light may be recorded in the form of a time correlation
function, the autocorrelation function G(.tau.), which relates the
scattered light intensity at a time t, I(t), to that a certain time .tau.
later, I (t+.tau.), as follows: G(.tau.)=<I(t)I(t+.tau.)>
where <> denotes averaging over all starting times t.
The photocurrent correlation function will have a form which in first order
can be expressed as consisting of two exponential functions:
G(.tau.)=(I.sub.f +I.sub.s).sup.2 +.alpha.(I.sub.f e.sup.-.GAMMA.
f.sup..tau. +I.sub.s e.sup.-.GAMMA. S.sup..tau.).sup.2. . (1)
In this equation, I.sub.f is the intensity of light scattered from the fast
diffusing protein species within the ocular tissue and I.sub.s is the
intensity of light scattered from the slow diffusing protein species in
the ocular tissue. .GAMMA..sub.f =D.sub.f K.sup.2 is the decay rate of the
fast diffusing species and .GAMMA..sub.s =D.sub.s K.sup.2 is the decay
rate of the slow diffusing species. D.sub.f and D.sub.s are the
diffusivities of the fast and slow species respectively.
K=(4.pi.n/.lambda.)sin(.theta./2) is the scattering vector where n=index
of refraction of the lens; .lambda.=wavelength of the laser in vacuo; and
.theta.=scattering angle. Additionally .alpha.=(1/2N) where N=number of
coherence areas in the exit aperture of the collection optics.
The first step in the analysis is to take the experimental measurements of
G(.tau.) and to fit it to the mathematical form represented in Eqn. 1.
This mathematical fitting procedure is carried out in the computer using
one of a number of well-established fitting routines. (It should be noted
that in general more than two exponentials may in fact be contained in
G(.tau.). However, because of the limited signal to noise ratio which
results from a clinically desirable short measurement time, approximately
(1-3 sec), the correlation function could be fit quite satisfactorily to
Eqn. 1.) As a result of the fitting procedure, one deduces the fundamental
parameters I.sub.f and I.sub.s and their sum I.sub.f +I.sub.s =I.sub.tot.
It is possible to show that I.sub.f and I.sub.s can be related to one
another using the following model for the origin of the slowly moving
component. The actual distribution of protein species in the lens may be
approximated as a simple bimodal distribution corresponding to a two state
model for the distribution of protein mass. Let N.sub.f denote the number
density (number per unit volume) of the small, fast diffusers, and let
N.sub.s denote the number density of the large aggregates. Furthermore,
let M.sub.f denote the molecular weight of the lighter species and M.sub.s
the molecular weight of the aggregates. Since the aggregates are composed
of the rapidly diffusing species, (M.sub.s /M.sub.f) is the number of
small proteins in each of the large aggregates. In a clear lens when no
aggregation has occurred N.sub.s =O, and all the protein is regarded as
existing in an unaggregated state. Under these conditions, the
corresponding number density of proteins is denoted as N.sub.f.sup.o. At
each point in the lens, the two state model of the formation of aggregates
implies the following condition:
N.sub.f.sup.o M.sub.f =N.sub.f M.sub.f +N.sub.s M.sub.s. (2)
This equation represents simply the conservation of mass. The left hand
side is the total mass density of the proteins associated with both peaks
of the biomodal mass distribution. N.sub.f M.sub.f is the mass density
associated with the light mass peak. N.sub.s M.sub.s is the mass density
associated with the heavy molecular weight peak in the mass distribution.
Since the protein concentration is known to vary spatially within the
lens, it may be expected that N.sub.f.sup.o will be a function of spatial
position inside the lens. In the normal young lens it is expected that
N.sub.s =O and N.sub.f =N.sub.f.sup.o. With aging and early
cataractogenesis, N.sub.f becomes smaller than N.sub.f.sup.O and N.sub.s
increases. If N.sub.f.sup.o is regarded as a constant independent of age,
then Eqn. (2) establishes at each time a relationship between N.sub.s and
N.sub.f based on the assumption that no new protein has been produced. Of
course, N.sub.f.sup.o can increase with age. This is to be expected
particularly in the cortex where protein expression continues with aging.
On the other hand N.sub.f.sup.o can be expected to be relatively
independent of age in the nucleus where no gene expression is occurring.
The parameters I.sub.f and I.sub.s may be expressed in terms of N.sub.f,
N.sub.s, M.sub.f and M.sub.s. Each of the scattering elements may be
regarded as being spatially distributed at random positions within the
illuminated region of the lens. Under these conditions, since the size of
the proteins of mass M.sub.f is approximately 100 .ANG. (i.e. small
compared to the wavelength of the light), I.sub.f is proportional to the
square of the mass (M.sub.f) of the scatterers and to the concentration
N.sub.f of scatterers. Thus
I.sub.f =k.sub.f N.sub.f M.sub.f.sup.2. (3a)
k.sub.f is a constant of proportionality independent of scattering angle,
but dependent upon such quantities as the incident light intensity and
polarization, the index of refraction of the protein compared to the mean
index of the lens, and the geometric arrangement of the light collection
optics in the QLS spectrometer.
In the case of the larger scattering element it is necessary to include the
fact that the size of these objects can be comparable to the light
wavelength. As a result, the intensity of light scattered from these will
be a strong function of the scattering angle. Thus, I.sub.s may be written
as:
I.sub.s (R.sub.s, .theta.)=k.sub.s (R.sub.s,.theta.)N.sub.s
M.sub.s.sup.2.(3b)
In this equation, k.sub.s (R.sub.s,.theta.), the coefficient of
proportionality, is expressly designated as dependent upon scattering
angle .theta., and the mean radius R.sub.s of the heavy scattering
element. Extensive calculations have been made regarding the form of
k.sub.s (R.sub.s,.theta.) for various shapes of dielectrics. It is
important to keep in mind that the angular anisotropy in the intensity of
light scattered from an aggregate whose size is comparable to the light
wavelength or larger can be quite large. Studies of this anisotropy factor
show that the ratio of the light intensity scattered near the forward
direction to that scattered towards the backward direction can range from
unity for small particles to approximately 100 for aggregates having sizes
of several thousand Angstroms. The scattering angle used experimentally
was approximately 135.degree.. Thus k.sub.s (R.sub.s, .theta.) could be as
much as 100 times smaller than k.sub.f if the scatterers are as large as
several thousand Angstroms in size. Of course, it is possible to select
scattering angles, in principle, anywhere within the back-scattering
quadrant, however, the choice of angle can significantly effect k.sub.s.
While the aggregates will vary in size from approximately 200 .ANG. to
approximately 1000 .ANG., it is only necessary to consider the largest of
these since they will have the greatest effect on scattering. This allows
a large amount of scattering to occur even when there are very few 1000
.ANG. aggregates present. Since this scattering may be detected, it is
possible to identify the existence of large aggregates even when there are
very few present.
Apart from these considerations, it is expected that k.sub.s will also
depend on light intensity and polarization, index of refraction and light
collection geometry in the same way as k.sub.f.
It is now possible to establish a relationship between I.sub.f and I.sub.s
(R.sub.s, .theta.), the experimentally measured parameters. The quantity
(N.sub.f.sup.o -N.sub.f) represents the number density of fast component
converted to aggregates. Both I.sub.f and I.sub.s can be expressed in
terms of this quantity. From equation (3a) it is found that:
I.sub.f =k.sub.f [N.sub.f.sup.o -(N.sub.f.sup.o -N.sub.f)]M.sub.f.sup.2,
thus
I.sub.f =I.sub.f.sup.o -k.sub.f M.sub.f.sup.2 (N.sub.f.sup.o -N.sub.f). (4)
Here I.sub.f .sup.o =k.sub.f N.sub.f.sup.o M.sub.f.sup.2 is the intensity
of light scattered from the fast diffusing species in the absence of any
aggregation. From Eqn. (3b) it is seen that I.sub.s is proportional
N.sub.s. Also, this quantity is proportional to (N.sub.f.sup.o -N.sub.f)
as follows from the conservation condition Eqn. (2):
N.sub.s =(N.sub.f.sup.o -N.sub.f) (M.sub.f /M.sub.s).
Using this in Eqn.(3b) gives:
I.sub.s (R.sub.s, .theta.)=k.sub.s (R.sub.s, .theta.)M.sub.s M.sub.f
(N.sub.f.sup.o -N.sub.f). (5)
Equations (4) and (5) express quantitatively the reduction in I.sub.f and
the increase in I.sub.s which is produced by the reduction (N.sub.f.sup.o
-N.sub.f) in fast diffusing species and their conversion to heavy
aggregates. This conversion gives a definite relationship between I.sub.f
and I.sub.s which can be obtained by expressing (N.sub.f.sup.o -N.sub.f)
in terms if I.sub.s using Eqn. (5) and substituting the results in Eqn.
(4) i.e.
##EQU1##
According to this result if I.sub.f and I.sub.s are measured in the nuclear
region where N.sub.f.sup.o remains roughly constant with age, as the
aggregation proceeds a plot of the values of I.sub.f and I.sub.s measured
at different times will produce a straight line with a negative slope
whose magnitude is S where
((k.sub.f M.sub.f)/k.sub.s (R.sub.s, .theta.)M.sub.s .tbd.S.(7)
The value of the intercept in this plot at I.sub.s =O is I.sub.f.sup.o. In
general, I.sub.f.sup.o can be expected to change with position in the
lens. In the cortex where protein is being produced I.sub.f.sup.o can be
expected to change with age as well as position. The size of the fast
particles can be estimated approximately 100 .ANG. while that of the large
scatterers is taken to be approximately 1000 .ANG.. The ratio (M.sub.f
M.sub.s) is approximately (1/10).sup.3 or 10.sup.-3. It is also possible
to estimate from the angular disymmetry of the large aggregate that
[k.sub.f /k.sub.s (.tau.)] is approximately 10.sup.2. Thus the quantity S
may be roughly estimated as 0.1.
Since I.sub.tot =I.sub.s +I.sub.f is the total intensity of light scattered
into the collection optics from the mobile scattering elements, it is
possible to examine how I.sub.tot depends upon I.sub.s and I.sub.f
separately:
(I.sub.tot)=I.sub.f +I.sub.s. (8)
Using Eqn (6) in the above yields:
##EQU2##
Thus a plot of I.sub.tot versus I.sub.s has an intercept at I.sub.s =O of
I.sub.f.sup.o, and a slope of (1-S). If the estimate of S as approximately
equal to 0.1 is correct, the slope of the I.sub.tot versus I.sub.s graph
will be somewhat less than unity. If measurements of I.sub.tot and I.sub.s
are made at a fixed position in the lens for an ensemble of subjects at
various ages and varying early stages of cataract development, the
corresponding pairs of points in a graph of I.sub.tot versus I.sub.s are
expected to fall on the straight line given by Eqn. (9). This will be true
if I.sub.f.sup.o is approximately the same for each subject, and if S
remains at approximately 0.1 or less for each subject. On such a
"universal curve" the positioning of a point along the vertical or
I.sub.tot axis indicates the degree of formation of heavy aggregate
species. If the measurements I.sub.s and I.sub.f indicate that I.sub. tot
is approximately 20 I.sub.f.sup.o, such a state of aggregation is likely
to produce a degree of turbidity quite apparent on visual observation. The
great advantage of the present form of investigation is that it provides a
linear and therefore, very sensitive measure of the earliest stages of the
aggregation process well before such aggregation could be detected using
either photographic or visual detection, through the slit lamp microscope.
The precise value of I.sub.tot or I.sub.s as a function of time during the
progression of the disease provides a very useful quantitative
characterization of the development of the aggregates.
It is also possible to relate the increase in I.sub.tot to the decrease in
If as follows: Since I.sub.tot =I.sub.f +I.sub.s, I.sub.s may be expressed
in terms of I.sub.f using Eqn. (4) and Eqn. (5). This results in the
following relationship between the measured quantities I.sub.f and
I.sub.tot :
I.sub.f =[1/(1-S)]I.sub.f.sup.o -[S/(1-S)]I.sub.tot. (10)
Thus a plot of the experimental values with I.sub.f as ordinate and
I.sub.tot as abscissa should yield a straight line whose intercept at
I.sub.tot =I.sub.f.sup.o is I.sub.f =I.sub.f.sup.o and whose slope is
negative with a magnitude S/(1-S). Also I.sub.f =0 when I.sub.tot =(1/S)
I.sub.f.sup.o Eqn. (10) represents quantitatively the decrease in I.sub.f
which results from the conversion of the lighter proteins into the heavy
aggregates. The total disappearance of all the lighter proteins occurs
when the total light intensity reaches a value equal to I.sub.f.sup.o /S
where S is the important parameter defined in Eqn. (7).
Experimental studies have demonstrated that the theory above does in fact
describe the experimentally observed relationship between I.sub.tot, and
I.sub.s or I.sub.tot and I.sub.f within experimental accuracy. The
experiments have provided a fundamentally important practical result shown
in FIGS. 4 and 5. These figures show that a plot of I.sub.tot versus
I.sub.s is in fact a straight line, regardless of the identity or age of
the subject or the location studied along the optic axis of the lens.
Thus, it is possible to label a plot of I.sub.tot versus I.sub.s as a
universal curve. This universal curve permits a very useful method for the
quantitative characterization of the degree of cataract. In an actual
measurement of the photocurrent fluctuation, one determines I.sub.f,
I.sub.s and I.sub.f +I.sub.s =I.sub.tot. One then locates the position of
this point on the universal curve. The advance of cataract corresponds to
movement of this point in a direction radially outward along the universal
curve. A reversal of the cataractogenic process corresponds to a movement
of the system point (I.sub.tot, I.sub.s) towards the origin. By
establishing the value of the intercept I.sub.tot (I.sub.s
=0)=I.sub.f.sup.o, it is possible to construct a universal curve whose
ordinate axis is (I.sub.tot /I.sub.f.sup.o) and whose abscissa is I.sub.s.
A lens region for which no aggregation has occurred corresponds to
(I.sub.tot /I.sub.f.sup.o)=1. A lens region where so much conversion to
the aggregated states has occurred so that opacification is visible
clinically corresponds to (I.sub.tot /I.sub.f.sup.o) equaling
approximately 30. The value of the ratio (I.sub.tot /I.sub.f.sup.o) is
thus a direct measure of the degree of cataractogenesis.
In view of the fact that the universal curve is a straight line with an
effective slope of unity, it becomes clear that only a single coordinate
is needed to unambiguously locate a point on the curve. Thus, an
experimental determination of the numerical value of the ordinate, namely
the quantity I.sub.tot /I.sub.f.sup.o, can provide by itself a detailed,
reproducible indication of the precise degree of cataract development. As
can be seen from the data in FIG. 4, the values of I.sub.tot
/I.sub.f.sup.o range from unity in the case of a perfectly normal lens
without cataract, to approximately 30 for a region in the lens exhibiting
a large degree of cataract formation.
This invention involves the elimination of the autocorrelator or spectrum
analyzer. More specifically, by measuring the intensity of light scattered
from the clearest part of a subject's lens (assumed to be I.sub.f.sup.O)
and by comparing this value with the intensity of light scattered from a
selected measurement location of the lens (I.sub.tot), it is possible
directly to determine the degree of cataractogenesis at the measurement
location. The greater the ratio of I.sub.tot /I.sub.f.sup.o, the greater
the number of aggregated, slowly diffusing proteins at the specific
location. This method is particularly advantageous because it provides
very accurate measurements with a minimum of complicated equipment. The
autocorrelator has been eliminated, as have a major share of the
calculations needed in conjunction with previous methods.
Alternatively, a calibrating element can be provided to preset the value of
I.sub.f.sup.o corresponding to a previously determined average value of
the light scattered from normal clear regions of the lens. This value of
I.sub.f.sup.o can be obtained as the result of statistical analysis of
data obtained from light scattered from normal, clear lenses. By providing
such a calibrating element for I.sub.f.sup.o, two objectives are achieved.
First, the necessity of accurately determining I.sub.f.sup.o for the
individual patient is obviated; and second, the resulting values of
I.sub.tot /I.sub.f.sup.o are standardized. This standardization enables
more reliable longitudinal studies of cataract development, as well as
intercomparison between different subjects and different measurement
locations in the lens. In the preferred embodiment, the calibrating
element comprises a suspension of polystyrene latex spheres in water, the
sphere size and concentration adjusted to allow the calibrating element to
have the same turbidity as a normal, clear lens.
Turning now to the Figures, FIG. 1 is a schematic representation of an
optical scattering analyzer for the study of the temporal fluctuations of
laser light scattered from the lens in vivo. In FIG. 1, a source of
substantially monochromatic, coherent, collimated light 10, such as a
laser, delivers a light beam 15, to the subjects eye 20, through a
delivery means, which may consist of, for example, a focusing lens 30
which couples the light into a fiber optic delivery cable 40, and a set of
delivery optics 50. The delivery optics serve to focus the light 15 onto
the subject's eye at the specific location at which the measurement is to
be taken. The light must be focussed for two reasons. First, the size of
the illuminated area is inversely proportional to the coherence area of
the scattered light. By focusing onto a small area, a greater coherence
area is obtained which allows easier measurement. Second, the incident
contact area on the lens in inversely proportional to the scattering area
on the retina. Thus by focusing down on the eye lens, the light going to
the retina is diffused, thereby preventing retinal damage. Scattered light
18 from the eye lens, passes through a collector such as an objective lens
60 onto a beam splitter 70. The beam splitter 70 serves to allow the
observer to focus and position the incident light while measuring the
scattered light. Part of the beam passes through the beam splitter 70 to
an ocular lens 80, which focuses the image for the observers eye 25. By
observing the scattering in this manner, a physician can control the laser
beam and position it at a specific location on the subjects eye 20. The
portion of the scattered light which is reflected by the beam splitter 70
passes into a collection means which can consist of a relay lens 90 to
focus the light reflected from the beam splitter 70, an aperture stop 100
to limit the length of the beam observed, a coherence angle aperture 110
to limit the number of coherence areas collected, a fiber optic collection
cable 120, and an optical filter 130 to filter out any extraneous light
from that to be measured. Light passing through the optical filter 130, is
converted into a photoelectric signal by an optical square law detector
140, such as a photomultiplier tube or solid-state photodiode. The signal
from the optical square law detector 140 is pre-processed by a
preamplifier and discriminator 150. This signal is then inputted into a
signal processor 160 which can be an autocorrelator or a power spectrum
analyzer and a computer 170 for processing as discussed previously. The
autocorrelation function or power spectrum and any calculated parameters
can be shown on a display 180.
FIG. 2 is a presentation of measurements of I.sub.f *, the intensity of
light scattered from the rapidly diffusing component, as a function of age
as measured in the anterior nucleus in twenty-nine subjects. I.sub.f * is
obtained from the computer analysis of the correlation function and is
directly proportional to I.sub.f. The numerical constant relating I.sub.f
* to I.sub.f is determined entirely by instrumental gain factors. This
graph shows clearly the statistically significant reduction in the
concentration of this unaggregated protein as the lens nucleus ages.
The rate of diffusion .GAMMA..sub.f this component was measured and found
to be 1.5.times.10.sup.4 sec.sup.-1 <.GAMMA..sub.f <2.5.times.10.sup.4
sec.sup.-1. Using these values of I.sub.f and the value
K=2.60.times.10.sup.5 cm.sup.-1, the diffusion coefficient D.sub.f is
found to be 2.2.times.10.sup.-7 cm.sup.2 /sec<D.sub.f <3.7.times.10.sup.-7
cm.sup.2 /sec. These values can be compared with the value
2.23.times.10.sup.-7 cm.sup.2 /sec found from light scattering studies on
monodisperse alpha crystallins. It is possible, therefore, to associate
the rapidly diffusing species with the unassociated .alpha.-crystallin
proteins in the lens cell cytoplasm. The decrease of I.sub.f * with age is
consistent with biochemical studies showing the gradual disappearance of
the alpha crystalline in the aging lens nucleus.
In FIG. 3, I.sub.tot versus age for 33 persons as measured in the lens
central nucleus is plotted. This graph shows that in the age group ranging
from 20 to 50 years of age the scattered intensity remains relatively
small. However, with aging beyond 60 years, a statistically significant
increase of total scattered intensity with age is observed. This increase
reflects age related development of senile nuclear cataract The results
presented in FIG. 3 are consistent with previous transmission studies done
by others This in-vivo data combined with the linear relationship between
I.sub.tot and I.sub.s shown in FIG. 5 indicates that the development of
senile cataract in the nucleus is associated with the conversion of the
rapidly diffusing crystallin species to much larger slowly diffusing
molecular aggregates.
FIG. 4 represents the results of measurements made on five patients
presenting preoperative lenticular opacification distributed at various
locations in the lens. All the data were obtained at or near the optic
axis of the lens. Typically five or six points ranging from anterior
cortex to posterior nucleus were studied for each patient. The patients
ranged in age from 48 to 71 years. I.sub.tot is plotted along the
ordinate. Also, a qualitative visual designation of the degree of
scattering associated with the observed values of I.sub.tot is indicated
along this axis. On the abscissa, I.sub.s * is plotted. I.sub.s.sup.* is
measured in the same units as I.sub.f * . As with I.sub.f *, the numerical
constant relating to I.sub.s * to I.sub.s is determined entirely by
instrumental gain factors. The entire range over which I.sub.tot is
measured corresponds to about a factor of 30 ranging from I.sub.tot of
approximately 0.2 when I.sub.s * is approximately 0 to I.sub.tot of
approximately 6 when I.sub.s * approximately 2.6. All measurements were
confined to points which did not exhibit the multiple scattering
associated with opaque regions. The range of I.sub.tot studied corresponds
to the full domain of early cataract development. This domain corresponds
to a factor of about 30 in total scattered intensity, as is consistent
with the previous discussion. It is also observed that in this domain of
scattering I.sub.tot is indeed linearly proportional to I.sub.s as
predicted in Eqn. (9). This linear relationship holds both for all
individuals studied, and for all the points studied in the lens of a
single individual. Thus, the 48 year old patient (indicated by X on the
Figure) has values for I.sub.tot ranging from I.sub.tot of approximately
0.9 to I.sub.tot of approximately 5.5. This is a quantitative expression
of the fact that along the optic axis of this patient's lens, there are
"normal" zones in which little conversion from fast to slowly diffusing
proteins has occurred Here 0.9.ltoreq.I.sub.tot .ltoreq.1.5. Nevertheless,
a few millimeters away, a considerable conversion to the slowly moving
species has occurred Here the light scattering is much stronger:
4.0.ltoreq.I.sub.tot 5.5. This graph clearly suggests that measurement of
I.sub.tot and I.sub.s at a fixed position in the lens can permit a
quantitative characterization of the degree of cataract development at
that position. As cataractogenesis proceeds at a fixed position in the
lens, I.sub.tot and I.sub.s along the "universal curve" of I.sub.tot
versus I.sub.s. On the other hand, the decrease of I.sub.tot and I.sub.s
along the universal curve would correspond to an increase in fast
diffusing species and a reduction the amount of slowly diffusing proteins.
The finding that all the data from each person and each location falls on
the I.sub.tot versus I.sub.s line clearly suggests that this "universal
curve" can provide a very sensitive, linear and quantitative
characterization of the degree of the molecular changes associated with
early cataract. It should be kept in mind that visual or photographic
detection of early cataract is characterized by a logarithmic response and
is consequently less sensitive particularly in the early stages of
cataractogenesis.
An investigation at fixed points in the lens of the slope (1-S) of the
linear relationship (Eqn. 9) between I.sub.tot and I.sub.s was also
obtained. In FIG. 5 a plot of I.sub.tot versus I.sub.s as observed at the
central nucleus for a subgroup of 28 patients with preoperative cataracts
in the age group from 23-82 years is shown. This subgroup was selected to
consist of individuals having no "opaque" or "transitional-opaque"
regions in the lens nucleus. In analyzing the correlation function in
these experiments the same units were chosen for the measurement of
I.sub.tot and I.sub.s. FIG. 5 shows unambiguously that in the central
nucleus the relationship between I.sub.tot and I.sub.s is quite linear.
From this graph it can be deduced that the magnitude of the slope
parameter [see Eqn. 7 and 9] is given by (1-S)=(0.96.+-.0.02).
I.sub.f.sup.o =0.055.+-.0.015 at the center of the lens nucleus.
Similar studies were conducted on the same population at the anterior
nucleus and the posterior nucleus. Table I below lists the measurements of
(1-S) and I.sub.f.sup.o at each of these positions. The units for the
determination of I.sub.s and I.sub.tot are the same and are determined by
gain factors in the autocorrelation output channels. Thus the absolute
magnitude of I.sub.f.sup.o as listed in Table 1 is without physical
significance. Nevertheless the magnitude of I.sub.f.sup.o sets the scale
against which cataract induced changes in I.sub.tot and I.sub.s are to be
compared. The slope (1-S) indicates that the important dimensionless
factor S as defined in Eqn. 7 has the value S=0.04.+-.0.02. This is
consistent with the previous order of magnitude estimate of 0.1 for the
value for S based on the assumption that the slowly diffusing corresponds
to an element roughly 1000 .ANG. in size.
TABLE 1
______________________________________
Measurements of the Slope and Intercept of
the Relationship I.sub.tot = I.sub.f .degree. +(1 - S)I.sub.s at
Various Positions in the Lens
Position Slope I.sub.f .degree.
______________________________________
Anterior Nucleus
0.96 .+-. 0.03
0.09 . | | |
