Title: Biological imager
Abstract: Apparatus and method for quantifying the biological components of a biological system having at least two different refractive indices through detection of wave front distortions. The biological component fractions are determined based on information gathered on their respective indices when exposed to particular wavelengths of light.
Patent Number: 7,006,219 Issued on 02/28/2006 to Prelewitz
| Inventors:
|
Prelewitz; David F. (Rochester, NY)
|
| Assignee:
|
Technology Innovations, LLC (West Henrietta, NY)
|
| Appl. No.:
|
441837 |
| Filed:
|
May 20, 2003 |
| Current U.S. Class: |
356/337; 356/338 |
| Current Intern'l Class: |
G01N 21/00 (20060101) |
| Field of Search: |
356/335-343,72,73,317,318,336
250/201.9,208.1
|
References Cited [Referenced By]
U.S. Patent Documents
Other References
Goodman, Joseph W., Frequency Analysis of Optical Imaging Systems, Introduction
to Fourier Optics, 1968, pp. 120-125, McGraw-Hill Book Company.
Fermeglia, Maurizio and Torriano Giovanni, Density, Viscosity, and Refractive
Index for Binary Systems of n-C16 and Four Nonlinear Alkanes at 298.15K, Journal
of Chemical Engineering Data, 1999, pp. 965-969, vol. 44, No. 5.
|
Primary Examiner: Stafira; Michael P.
Attorney, Agent or Firm: Salai, Esq.; Stephen B., Ryan; Thomas B., Harter, Secrest & Emery LLP
Claims
The invention claimed is:
1. A method of determining the amounts of first and second non-immiscible components
in a mixture having known, different indices of refraction in the mixture comprising:
a) passing a focused light beam through the mixture;
b) measuring the displacement of the actual point of focus from a known focal
point through a material with a known index of refraction; and
c) calculating the amounts of the first and second components present from the displacement.
2. The method of determining the amounts of the non-immiscible components present
in a mixture as set forth in claim 1, comprising:
a) passing a focused light beam having at least two discrete wavelengths through
the mixture; and
b) separately measuring the displacement of the actual point of focus from a
reference focal point for each of said at least two discrete wavelengths.
3. The method of determining the amounts of biological components present as
set forth in claim 1, comprising:
filtering the emergent light beam to remove higher order distortions.
4. The method of determining the amounts of biological components present as
set forth in claim 1, comprising:
collimating the beam after it passes through the biological components and before
measuring the displacement of the focal point.
5. The method of determining the amounts of biological components as set forth
in claim 4, in which measuring the displacement comprises measuring the shape of
a collimated wavefront beyond the point of focus.
6. A method for measuring characteristics of a biological system having at least
two contrasting density components comprising:
(a) projecting light of wavelength λ
1 through a multi-density
system to distort wavefront of wavelength λ
1;
(b) projecting light of wavelength λ
2 through a multi-density;
(c) system to distort a wavefront of wavelength λ
2; and
(d) determining biological components information in response to the distortion
of the light of wavelengths λ
1 and λ
2,
wherein the biological information determines the refractive index of a biological component.
7. A method for measuring characteristics of a biological system having at least
two contrasting density components comprising:
(a) projecting light of wavelength λ
1 through a multi-density
system to distort wavefront of wavelength λ
1;
(b) projecting light of wavelength λ
2 through a multi-density;
(c) system to distort a wavefront of wavelength λ
2; and
(d) determining biological components information in response to the distortion
of the light of wavelengths λ
1 and λ
2,
wherein the biological information determines the ratio of biological components.
8. A method for measuring characteristics of a biological system having at least
two contrasting density components comprising:
(a) projecting light of wavelength λ
1 through a multi-density
system to distort wavefront of wavelength λ
1;
(b) projecting light of wavelength λ
2 through a multi-density;
(c) system to distort a wavefront of wavelength λ
2; and
(d) determining biological components information in response to the distortion
of the light of wavelengths λ
1 and λ
2,
wherein the biological information determines both the refractive index and the
ratio of biological components.
9. A method of analyzing biological components in a system, having a plurality
of contrasting density components comprising:
a) a projecting light comprising λ
1 and λ
2 through
the fluid stream such that
(i) λ
1: chosen such that N
BC (λ
1)≠N
water
(λ
1) and N
BC (λ
1)≠N
air (λ
1);
(ii) λ
2: chosen such that N
water (λ
2)≠N
water
(λ
2) and N
BC (λ
2)≠N
air (λ
2);
b) projecting light through the system such that displacement of the focus from
a known position indicates a specific biological component;
c) further projecting light through an aperture and collimating lens array to
an area sensor; and
d) detecting the wavefront shape distortions to determine the specific biological components.
10. The claim of claim 9, further comprising using a strobe to determine movement
of the components.
11. A method of analyzing biological components in a system, having a plurality
of contrasting density components comprising:
a) a projecting light comprising λ
1 and λ
2 through
the fluid stream such that
(i) λ
1: chosen such that N
BC (λ
1)≠N
water
(λ
1) and N
BC (λ
1)≠N
air (λ
1);
(ii) λ
2: chosen such that N
water (λ
2)≠N
water
(λ
2) and N
BC (λ
2)≠N
air (λ
2);
b) projecting light through the system such that the focus indicates a specific
biological component;
c) further projecting light through an aperture and collimating lens array to
an area sensor; and
d) detecting the wavefront distortions to determine the specific biological components,
further comprising tuning the detector to detect a plane wave when projected
through water.
12. A biological imager for measuring the biological components in a system comprising:
a) a light source comprising wavelengths λ
1 and λ
2
such that when projected through the biological sample, produces a distinct
signature due to the distortion of the wavefront; and
b) detection to sense the signature,
wherein a focus moves left or right to indicate more or less of a component.
13. The claim of claim 12, further comprising using a Shack-Hartmann Wavefront Analyzer.
14. The claim of claim 12, further comprising solving the first order differential equations.
Description
BACKGROUND OF THE INVENTION
1. Filed of the Invention
This invention relates to an apparatus and method for analyzing different components
in a system having at least two different refractive indices. This invention further,
and more particularly, relates to biological imaging through the components of
a biological system.
2. Background Art
In many situations the monitoring of a biological system in real time is desired
in addition to determining the biological components without using a computer-intensive technique.
It is desirable to make measurements without having to recalibrate each time a
measurement is taken, such as those that use absorption techniques. Different material
properties require recalibration of currently used equipment. This invention addresses
this problem.
BRIEF SUMMARY OF THE INVENTION
This invention relates to an apparatus and method for analyzing a system using
the refractive index of light. The biological component fractions of a biological
system are determined using the refractive index of materials in relation to specific
wavelengths of light.
The invention can determine the percentages of biological components and water
without prior knowledge of the refractive index of the biological components. The
method determines the percentages of biological component fractions, include passing
a focused light beam through the biological components, measuring the displacement
of the point of focus from a known focal point with a known index of refraction,
and thereby calculating the percentages of biological components present.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
FIG. 1
a is a diagram of the biological imager of this invention.
FIG. 1
b is a diagram of another biological imager.
FIGS. 2 and 2
a show schematic diagrams of the biological imager.
FIGS. 3
a-3
c show schematic diagrams of the biological imager.
FIG. 4 is a schematic diagram of one embodiment of the biological imager with
an area sensor and lens array.
FIG. 5
a is a schematic of the biological imager with a reference fluid.
FIG. 5
b is a schematic of the biological imager with the reference material
and another lighter fluid.
FIG. 5
c is a schematic drawing of the biological imager with the reference
material and another heavier fluid.
FIG. 5
d is a schematic of the biological imager with the reference material
and with both a lighter and a heavier fluid.
FIG. 5
e is a schematic drawing of the biological imager and a Shack-Hartmann detector.
FIG. 6 is an embodiment with an imaging lens.
FIG. 7 shows a schematic drawing of the strobe and sensor arrangement.
DETAILED DESCRIPTION OF THE INVENTION
Virtually every biological system consists of a variety of components composed
of fluids and gels that exist as a mixture, each component with one or more distinct
refractive indices when a specific wavelength of light passes through the mixture.
Typically elaborate imaging methods must be used to image these biological systems
and to measure their physical properties such as viscosity, geometry, relative
fractions, and flow rates if needed.
Fluids and gels, specifically those of different biological components, refract
light by varying degrees when a specific wavelength passes through the mixture.
The amount of refraction is a function of fluid composition and wavelength of the
light passing through the fluid. The refractive index is a physical property of
the fluid and is a parameter for determining the optical interaction of the fluid
and the light refracted through it.
This invention is applicable to all systems including biological systems. For
purposes of brevity, however, the description herein will be primarily directed
to invitro biological systems, particularly a cell with components composed of
protein matrix-based gels.
FIG. 1
a shows a biological imager
10 deployed to analyze a biological
mixture of biological components including water, particulate matter, and other
materials that could be found in a biological system. FIG. 1 shows a cell
11
with a variety of components such as protein, matrix-based gels hereafter referred
to as a biological mixture
12 which could be any system of components but
is shown here as a cell. Control of the fluid of the bath, as well as its movement
if relevant, is known and can be described in a variety of ways, some of which
are not to be discussed in this application.
The biological imager
10 has a light source
14 and a detector
16
arranged on opposite sides of a sample of a biological mixture
12 which
is made up of non-immiscible biological components. This mixture must be such that
when separated it retains its ratio. The biological imager is such that there are
transparent, or partially transparent, openings
18 between the light source
and the detector that allow light to pass from the light source through the biological
mixture
12 to detector
16. The biological imager
10 can incorporate
any number of optical elements, including but certainly not limited to lenses,
filters, diffraction gratings, and other optical elements that will be discussed
in detail later. These optical elements can be incorporated into the openings
18
or can stand alone.
The light source
14 is a point source or extended point source with one
or more discrete wavelengths temporally and/or spatially separated such as would
be true for a single source that is pulsed or one or more spatially separated sources.
The source can include one or more discrete wavelengths or be a filtered white
light source. If there are two or more light sources they can have overlapping
spectra but the wavelengths must be at least detectable so that there is sufficient
energy that is unique to each wavelength to provide two unique refractive properties
after the light has passed through the fluid mixture. Note that alternatively a
wideband white light source could be used unfiltered (without discrete wavelengths
detectable at the source) and filtered at the detector. What is required is that
the two wavelengths must be discrete to provide distinct and separate information
when separately focused. Each discrete wavelength will be separately focused and
the shift in the focal point measured from a known focal point.
FIG. 1
b shows a biological imager
10a to analyze the fluid
mixture
12 where the detector
16 is in an alternate location. The
biological imager
10a has a second surface
19 that can incorporate
the detector
16 or may be reflective or partially reflective such that the
detection of a component may be directly read, recorded on the surface
19
or reflected toward another location. This embodiment could incorporate a circuit
that diverted the focal point electronically as could the other embodiments.
FIGS. 2 and 2
a are detailed schematic diagrams of the biological imager
10 shown in a container
20 which could be a laboratory. The fluid
mixture
12 is shown between the light source
14 and the detector
16. In this embodiment there is a first quadradric phase plate (L
1)
22 and a second quadradric phase plate (L
2)
24 both of
which preferably are positive lenses, and hereafter referred to as first lens
22
and second lens
24. Light from the source
14 can be focused in the
fluid mixture
12 where a real image (I
1) of the source
14
is formed by L
1. The light travels on to (L
2) which can form
another image (I
2) near an aperture or spatial filter
26 before
being focused by a third collimating lens
28 onto the lens array
30
and an area sensor
32 which could be a focal plane array. It is not necessary
that the focus occur in the fluid mixture
12. The volume of the fluid mixture
12 that is being analyzed will be referred as the analysis zone
34
in the following discussion. The analysis zone is also referred to as a capturing
cone. The fact that this covers a larger volume allows integration and averaging
of a larger volume of fluid mixture
12.
FIG. 3
a; FIG. 3
b, and FIG. 3
c, show alternate arrangements
of a light source
14 and the detector
16 as well as one or more lenses
that would work under certain circumstances. FIG. 3
a has the first lens
22, the aperture
26, and the collimating lens
28. FIG. 3
b
does not have the collimating lens
28 and so the detector
16
must be able to handle light that has not been collimated. In this scenario, it
may be more difficult to determine a unique solution due to the presence of higher
order distortions. The same would be true if the collimating lens
28 was
present but the aperture
26 was removed. The aperture
26 is not required
in certain circumstances. FIG. 3
c adds a filter
35 so that a white
light source can be used without a filter at the source but with some sort of filter
at the detector
16. The detector filter could even be an electronic device
or involve an algorithm.
FIG. 4 shows the light source
14 directed toward the first collimating
lens (L
1)
22 which in this case is shown to be at a distance
that is twice its focal length from the light source. The first lens could be any
distance from the light source as would be known in the art as long as the expanding
wave front is known as it enters the biological mixture
12. The wave front
will be refracted by the first lens
22, refracted through the biological
mixture
12, and in this embodiment, if refracted through pure water, would
focus at a point
36 between the first lens
22 and the second lens
24. The focus point
36, if it was pure water, would be N
water
(refractive index of water)·2·f
1 (focal length of the first
lens
22) from the first lens
22, and a distance equal to N
water
(refractive index of water)·2·f
2 (focal length of the
second lens
24) from the second lens
24. The lenses
22 and
24 are separated by a distance "d" shown by
38. The emerging light
would be refracted by the second lens
24 and directed toward the spatial
filter
26, which in this embodiment is a distance equal to 2·f
2
from the second lens
24. After passing through the biological imager
10 the light wave front has been distorted by scattering in the fluid. The
distorted wave front represented by
40 in the diagram would defocus by higher
order terms incorporated in it, as shown in the diagram by the wavy line
42.
After this distorted wave front
40 passes through the spatial filter or
aperture
26, the wave front has some of the noise eliminated leaving biological
fluid mixture dependent defocus. The choice of an aperture or spatial filter
26
is critical to the success of this apparatus because, like a confocal microscope,
it eliminates noise (higher order distortions) without removing the focus information.
If the aperture is too small, the information that includes the mixture dependent
focus would be lost; but if the aperture is too large, unnecessary noise would
detract from the efficiency of the apparatus. All of the distances must be measured
precisely since the shift in the focal point will be the order of a wavelength.
The filter aperture requirements (size, geometry, etc.) are heavily dependent
on the optical system layout and the defined measurement tolerances. Given that
defocus shifts are the primary wavefront aberration to be measured, all other contributions
to the WFE (wavefront error) can be ignored. The filter aperture
26 can
help reduce the other aberrations (typically, of a higher order than defocus),
which are primarily due to scattering generated by the material being measured.
A basic review of how to deal with such things can be found in Goodman's book "Introduction
to Fourier Optics", in chapter and section: "Frequency analysis of optical imaging
systems, Aberrations and their effects on frequency response" (Chapter 6-4 in the
1
st edition). Here, the generalized exit pupil function is defined as:
P(
xp,yp)=
p(
xp,yp)exp(
jkW(
xp,yp)),
where
p(
xp,yp)
is the non-aberrated pupil function applied to the image at aperture
26.
W(x
p,y
p) encompasses the aberration phase terms of the exit
pupil wavefront. Assuming defocus is the dominant term we have:
##EQU1##
where ε is the phase error term. The specified shifts in defocus are
related to ε and an aperture
26 can be constructed such that the higher
order contributions are minimized with respect to the desired measurable defocus range.
In this embodiment the third collimating lens
28 (also referred to as
"a
fourier transform lens" or "FT lens") is placed a distance equal to its focal length
from the spatial filter
26. The third, collimating lens
28 essentially
turns the wave front "inside out" and the focus information is the largest component
of the light wavefront leaving the collimating lens
28. The light is focused
on the lens array
30 of this embodiment which could take many different
formats (such as Shack-Hartmann, Interferometry phase diversity, various algorithms,
electric circuits, etc.). A Shack-Hartmann area sensor
32 can perform inverse
fourier transform resulting in spot shifts when a refractive index of the biological
mixture
12 changes. If the parameters are carefully chosen and tuned so
that there is no shift when the medium is water, and there is a positive shift
when there is the presence of certain components and there is a negative shift
when there is the presence of other components allowing a simple deflection measurement
to determine the fraction of certain components in a sample. The area sensor
32
could take another format such as interferometer, which would require the transmission
of an undistorted wavefront from the light source
14 to the detector
16
to the area sensor
32 in order to get the interference necessary for the
interferometer to work. In which case, there would be no need for the collimating
lens
28.
FIG. 5
a is a schematic diagram of the biological imager
10 and
a reference fluid with a known refractive index such as water, calibrated so that
the focus of the light passed through at the detector
16.
FIG. 5
b is a schematic drawing of the biological imager
10 and
both the reference fluid and another lighter fluid such that the focal point changes
in relation to the change in refractive index due to the amount of biological components
in the mixture.
FIG. 5
c is a schematic drawing of the biological imager
10 and
both the reference fluid and another heavier fluid such that the focal point changes
in relation to the change in refraction index due to the heavier fluid. Note that
the focal point will shift in a direction opposite of that in FIG. 4
b in
this example. The introduction of the lighter gas causes less refraction because
the light is traveling through a fluid with a lower refractive index.
FIG. 5
d is a schematic drawing of the biological imager
10 and
the reference fluid, as well as both a lighter and a heavier fluid so that there
is the need to focus two different wavelengths of light to solve for the two unknown
fractions of biological components present.
FIG. 5
e is a schematic drawing of the biological imager
10 with
all three phases of fluid and a Shack-Hartmann detector.
The biological imager
10 of FIG. 6 is set to analyze a fluid flow of a
biological mixture. This biological system could be part of an organism. A sample
from an organism, or it could be in a separate vessel or system in a laboratory.
This is particularly effective in organisms because of the gel-like nature of living
cytoplasm, the interior living cells. Many cellular functions can be attributed
to and are accomplished by gel properties of sub-membrane cytoskeleton or actin,
microtubules and other protein structures such as regulating ionic fluxes and concentrations.
Cytoplasmic gels manifest collective phase transitions such as polymerization of
actin proteins with accompanying ordering of cell water and exclusion of large
cations. These collective phase transitions can explain not only ionic fluxes,
but also voltage gradients, propagating action potentials, mitosis, muscle contraction
and cell movement. The fact that cells include and are regulated by components
such as the protein matrix-based gels make this invention particularly useful.
Since the cytoplasm is intrinsically reactive and able to maintain cell homeostasis
and functions, the cytoplasm gel best captures the essence of the living state
and can be measured in response to the refractive index of light in accordance
with this invention.
FIG. 7 shows a fluid stream that can be analyzed using this invention to determine
the rate of flow.
Any properties that can be derived from the different refractive indices of the
cellular components, particularly protein matrix-based gels. Properties such as
the geometry of the structure in a bath of known refractive index using the refractive
index of light is possible because the structure will refract light at the boundaries.
One embodiment of the method for measuring the biological fractions includes
projecting two discrete wavelengths λ
1 and λ
2 through
the biological components causing wavefront distortion allowing for the determination
of two separate focal point displacements and the determination of two biological
fractions in response to the measurements generated by λ
1 and
λ
2. This method requires values of λ
1 and λ
2
such that:
- (a) λ1: chosen such that NBC (λ1)≠Nwater
(λ1); NBC (λ1)≠Nair
(λ1); and
- (b) λ2: chosen such that NBC (λ2)≠Nwater
(λ2); NBC (λ2)≠Nair (λ2).
In order to solve for one unknown, for example the fraction of DNA, the following
equation is solved where:
- (a) OPL=Optical Path Length (measured by the refractometer)
- (b) OPL[measured]=Navg (λ);
- (c) ANBC1(λ1)+BNBC2(λ1)+CNBC3(λ1)=Navg(λ1);
- (d ANBC1(λ2)+BNBC2(λ2)+CNBC3λ2)=Navg(λ2);
- (e) A+B+C=1
- (f) N(λ)=refractive index.
In order for these equations to be solved, it is necessary that the fluid components
do not chemically interact. This is characterized by being able to be separated
with the component ratios preserved. In one example, when pure water is a reference
point, the focal point changes as a function of the material in the flow stream.
The light beam will curve (spread) when compared to the reference. This curvature
can be measured. There are a number of combinations that can be solved for including
a component refractive index or the ratio of components. If there are two or more
unknowns then additional wavelengths like λ
1 and λ
2
will be required to solve for these additional unknowns.
First the first λ
1 is focused and the distance from the known
focus in water measured so that N
avg (λ
1) can be calculated.
Subsequently, the second λ
2 is focused, the distance from the
known focus measured, and N
avg (λ
2) calculated. With
all but A, B, and C known the coefficients A, B, and C can be calculated from the
three equations.
If the refractive index of one biological component (N
BC2) is also
unknown but the refractive indices of two other biological components (N
BC1
and N
BC3), are known, then there are four unknowns (A, B, C and N
BC2)
since only N
BC1 and N
BC3 are known. To solve these equations,
four wavelengths (λ
1, λ
2; λ
3,
λ
4) must be focused and the distance from a known focal point
measured for each [N
avg (λ
1); N
avg (λ
2),
N
avg (λ
3), N
avg (λ
4)].
The N
BC2 varies in a known way according to the Cauchy relationship
such that:
- N(λ1)∝K1+K2N(λ12),
where the higher order terms are ignored, and then Noil, A, B, and C
can be solved for simultaneously. Including additional terms in the Cauchy expansion
will require additional wavelengths in order to find a solution.
A detailed analysis using the physical arrangement shown in FIG. 2 follows:
- a) Dimension Items
- (i) so1—Distance of light source (14) to
the 1st principle plane of Lens 1 (22)
- (ii) si1(Nave(λ))—Distance of
imaged light source (I1) to the 2nd principle plane of Lens
1 (22) for Nave (λ)
- (iii) so2(Nave(λ))—Distance of
imaged light source (I1) to 1st principle plane of Lens 2
(24) for Nave(λ)
- (iv) si1ref(Nref(λref))—Reference
distance of imaged light source (I1) to 2nd principle plane
of Lens 1 (22) for Nref(λref)
- (v) so2ref(Nref(λref))—Reference
distance of imaged light source (I1) to 1st principle plane
of Lens 2 (24) for Nref(λref)
- (vi) Δsi1(λ)—Change in si1
relative to reference at si2ref due to wavelength and material changes
between Lens 1 (22) and Lens 2 (24)
- (vii) d—Thickness of the material to be analyzed
- (viii) si2(Nave(λ))—Distance of
imaged light source (12) to the 2nd principle plane of Lens 2
(24) for Nave(λ)
- (ix) si2ref(Nref(λref))—Reference
distance of imaged light source (I2) to 2nd principle plane
of Lens 2 (24) for Nref(λref)
- (x) Δsi2(Nave(λ))—Change
in si2 relative to reference at si2ref due to wavelength
and material changes between Lens 1 (22) and Lens 2 (24)
- (xi) f3—Effective focal length of Lens 3 (28)
- (xii) α—Aperture size
- (xiii) so3(Nave(λ))13 Distance
of imaged light source (12) to 1st principle plane of Lens 3
(28) for Nave(λ)
- (xiv) z—Distance from aperture 26 to where the WFE
(wavefront error) is measured
- (xv) y—Distance perpendicular from optical center line to
where the WFE is measured
- (xvi) WFE(λ)—Paraxial Wavefront Error (measured in waves
of λref) relative to reference due to wavelength and material
changes between Lens 1 (22) and Lens 2 (24)
- b) Glossary:
- (i) λ≡Wavelength
- (ii) λref≡Reference wavelength
- (iii) N≡Refractive index
- (iV) f≡Effective focal length for all λ to be used
in device, where f>0 for all lenses
- (v) Nref(λref)≡Index of a reference
component (m=0) at a reference wavelength (λref)
- (vi) Am≡Solution component volume percentage
- (vii) n≡Number of solution components
- c) Known Terms:
- (i) λref, Nref(λref),
f1, f2, f3, so1, and d
- d) Equations:
##EQU2##
- (v) so3(λ)=f3-Δsi2(λ)
##EQU3##
- e) Number Run:
- (i) so1=100 mm
- (ii) λref=1.4 μm
- (iii) d=260 mm
- (iv) y=10 mm
- (v) z=50 mm
- (vi) f1=f2=f3=50 mm
- (vii) N0(λref)=1.3
- (viii) Nave(λ)=1.302
- (ix) Δsi2(Nave(λ))=0.03691172
mm; ∴
Most wavefront sensors can easily measure errors to less than 1 wave, and given
a small change of index, there is typically a significant change in the wavefront
error produced. For the above case, where there is a 0.002 index change, the WFE
is easily measurable.
Other properties that can be calculated include any physical property that
has a relationship that changes with the refractive index. The refractive index
relates to the interaction of light with the electrons in a substance, the more
electrons, and the more polarizable the electrons, the higher the refractive index.
Although viscosity is resistant to the shearing force, it is related to the interactions
between molecules as they move past one another. It is possible to relate viscosity
and other properties to the refractive index of light within a specific class of
components, specifically proteins for example, by correlating the two properties
and using the relationship. For example, for proteins, the viscosity increases
because there is more opportunity for them to interact as they are moving past
each other, and the refractive index also increases slightly because the density
of electrons is a little higher. For this very restricted class, a correlation
can be made that is valid for that class of proteins. Similar correlations could
be made for other non-immiscible components.
In order for these equations to be solved, it is necessary that the fluid components
do not chemically interact, such that the biological component may be separated
with the component ratios preserved. For example, when pure water is a reference
fluid, the focal point changes as a function of the material in the flow stream.
The light beam will curve (spread) when compared to the reference fluid. This curvature
can be measured. There are a number of combinations that can be solved including
a refractive index or a ratio of non-immiscible biological component fractions.
If there are two or more unknowns then additional wavelengths will be required
to solve for the unknown.
The Shack-Hartmann Wavefront Analyzer is constructed by placing an array of apertures
in front of a charge-coupled device or CCD camera. These apertures allow light
be diffracted by the plate onto the CCD. The segments of the beam that pass through
the apertures will be spatially displaced from the center position, based on the
direction of travel, or the phase of that part of the beam. The CCD camera measures
the phase of each spot by measuring this displacement. Software algorithms then
reconstruct a wavefront for the entire beam. The spacing of the apertures defines
the resolution of the system, and the size of each aperture is calculated to optimize
sensitivity to phase changes. In contrast, a Shack-Hartmann Wavefront Analyzer
uses an array of small lenslets to collect all of the beam in each aperture position,
and project all of it onto a detector.
Essentially, a spherical wavefront is refracted through the biological
mixture
12, which will eventually be focused. It is preferred that the focus
be located within the biological mixture
12. A key component is the aperture
or spatial fluid
26 which eliminates the majority of the (waste) scattered
light outside of the focus region. The aperture or spatial filter
26 functions
as a noise filter. This is how confocal microscopy works. Additionally, the aperture
size is optimized to account for focus shifts (+ or -) due to average volume index
changes. Any wavefront can be propagated through the test region, if the wavefront
is pre-determined before being transmitted through the distortion zone (e.g., a
component-water mix), and if there is a reference volume of material (e.g., water)
to make a comparison with. A distortion dependent shift in focus (defocus) is going
to be the largest distortion component, hence, the easiest to detect and measure
(even in a noisy environment).
Not only can the refractive index or relative fractions of components be calculated
but other relative functions like thickness, size, geometry, and viscosity of the
cellular components such as different fluids or gels such as the protein matrix-based gels.
Concerning a flow rate measurement method, a strobe will be used as shown
in FIG. 7 and accommodations made for the boundary effects in the container or
flow tube. The flow profile can be compensated by taking the flow rate at the center
of the container or flow tube and at the edges and averaging, or testing at the
center. LED's are strobed at different duty cycles until particles appear stationary
(within a certain tolerance). Hence, the velocity of the fluid can be determined.
The sensing array can have a central imaging lens to detect the flow rate and wavefront
sensor lenslets to detect the wave front information and distortions. With a fixed
imaging optic, the device measures the velocity of particulate matter in the focus
region in the fluid using a strobe. If the fluid ratios and component values are
known, the volume fluid flow rate can be calculated if the center flow rate has
been determined. By varying the gate time of the strobe, imaged particles may appear
stationary once the gate time is correct.
With a fixed imaging optic, the device measures velocity of particulate matter
in the focus region in the fluid using a strobe. With knowledge of the fluid ratios,
and density values, the fluid volume flow can be determined. It is also possible
to scan the imaging optic (using a speaker coil mounted optic as used in CD players)
and collect a range of flow data.
A number of basic improvements result, which include:
- a) reduction of errors due to optical scattering losses;
- b) simplification of instrument calibration;
- c) improved accuracy for low-water-cut (higher ratio of biological component
to water).
- d) elimination of calibration step;
- e) accurate multi-component detection system over all ratios; and
- f) flow measurements (if required).
While the invention has been described in connection with a presently preferred
embodiment thereof, those skilled in the art will recognize that many modifications
and changes can be made therein without departing from the true spirit and scope
of the invention, which accordingly is intended to be defined solely by the appended claims.
*
