 |
Description  |
|
|
BACKGROUND OF THE INVENTION
The present invention relates to methods and systems for directional,
enhanced fluorescence from molecular layers, including its use in
determining the presence or absence of a substance with a fluorescent
component.
The invention is an improvement on the fluorescent assay method and
apparatus described in U.S. Pat. No. 4,649,280, which issued Mar. 10, 1987
to Holland and Hall. Fluorescent assay systems typically employ optical
detectors to detect the light emitted from a fluorescing substance. When
light of a wavelength known to excite the fluorescent component of a
particular substance is incident on an unknown sample, the presence (or
absence) of the substance in the sample may be indicated by detecting the
presence (or absence) of a fluorescent emission. Well known to the art is
that the excitation and emission wavelengths of a fluorescent material are
different.
Any methods of chemical analysis, including fluorescent assay systems, are
improved by increasing the reliability of their results. U.S. Pat. No.
4,649,280 describes a method which increases the intensity of the
fluorescent light emitted from the sample. This increase decreases the
sensitivity required in the optical detector to determine the presence of
a substance, which allows the detection of smaller quantity of a substance
in a sample.
The method and apparatus described in the patent increased the light
intensity of a fluorescing substance by employing an optical waveguide to
generate a strong electromagnetic field in the vicinity of a film of
fluorescent material, and particularly from the layers of molecules of the
material attached to a wall of the optical waveguide. The excitation
radiation which was incident on the fluorescent material was self-coupled
to the waveguide to support the propagation of the modes which generated
the strong field. This combination caused more intense fluorescence (i.e.,
intensity of the fluorescence) relative to that excited by radiation
incident on conventional systems (i.e., fluorescent material coated on a
glass slide). The strong field generated by the propagating modes was
responsible for the increased fluorescence, and the increase was a
function of the dimensions of the waveguide which supported the waveguide
modes. The intensity of the fluorescence could be increased nearly 200
times that of conventional systems.
A number of problems were presented with the prior art systems.
The first problem was that the fluorescence emitted at a given wavelength
from conventional systems and the system described in U.S. Pat. No.
4,649,280 was diffusely distributed about a normal to the flat surface of
the fluorescent material. The result of this diffuse distribution was that
most of the fluorescence emitted by the sample was not collected by the
photodetectors. This diffuse distribution was compensated to some extent
by the increased enhancement from the system described in U.S. Pat. No.
4,649,280; however the basic inefficient distribution for detection
purposes remained. Since only a fraction of the fluorescence was detected,
the prior art measurement techniques and systems were inherently less
efficient than one that can make use of most or all of the fluorescence at
a given wavelength, as provided by the present invention.
Another problem with conventional systems and the improved system of U.S.
Pat. No. 4,649,280 was that they only determined the presence, absence
and, to a limited degree, the concentration of a material in a sample.
Also absent from these systems were other tests which would further
indicate the presence or absence of a fluorescent material in a sample.
Finally, if the fluorescent test was inconclusive, or if a check of the
results was desired to increase the conclusiveness of the analysis, a
completely separate method of analysis had to be used. This added expense
to the analysis and was time consuming.
W. R. Holland et al., Optics Letters, Vol. 10, No. 8, pp. 414-416 (August
1985) also describes waveguide mode enhancement of molecular fluorescence,
as described in the aforementioned patent, and ascribes the enhancement to
near field interaction between the fluorescent molecules and the waveguide
modal fields. A. M. Glass et al., Optics Letters, Vol. 5, No. 9, pp.
368-370 (September 1980), also reports the enhancement of fluorescent
material deposited on a silver film.
SUMMARY OF THE INVENTION
The present invention provides a method and system for directional,
enhanced fluorescence from molecular layers which simultaneously or
consecutively enables a multiplicity of tests to be performed to determine
the presence, absence or concentration of a material in a sample.
Another objective of the present invention is to provide a method and
system for directional, enhanced fluorescence from molecular layers which
detects substantially all the fluorescent radiation at a given wavelength.
Another objective of the present invention is to accomplish the
aforementioned objectives in a relatively simple and inexpensive manner.
Utilizing the method and system of the present invention, the fluorescent
light emitted at a given wavelength is radiated at discrete angles with
respect to the normal to the surface of the material. These discrete
angles of emission enable photodetectors to be arranged to detect most of
the fluorescent emission. The emission angles are a function of the
wavelengths of the emitted light as well as the state of polarization of
the emitted light and the waveguide thickness. The signal detected by the
present invention can have an enhancement factor close to 2000. The
"enhancement factor" is defined as the fluorescent intensity collected
from the examined system divided by the fluorescent signal collected from
a conventional system. The combination of directional emission, such
distinct angles having a functional relationship to wavelength,
polarization and thickness, as well as increased enhancement, allows a
number of features of a fluorescent emission to be analyzed simultaneously
and with a minimum of hardware, leading to more accurate results in a
simpler and less time consuming manner.
In general, the present invention relates to the method of improving the
detection, identification, and enhancement of fluorescence of a material
comprising the steps of:
(a) depositing a layer of the material on a waveguide with corrugated
surface(s) which supports propagation modes for optical radiation at the
wavelengths of absorption by and fluorescence from the material,
(b) exciting the fluorescence of the material through the waveguide
propagation modes at the wavelength of absorption by said material and at
discrete angles of emission to the normal to its surface, and
(c) detecting the fluorescence at said discrete angles from the film's
normal.
In accordance with the present invention, the basic structure of the
waveguide mode enhancement of molecular fluorescence system is modified by
adding corrugations to the waveguide structure. In existing waveguide mode
enhancement systems the modes of an optical waveguide are used to generate
a strong electronmagnetic field in the vicinity of a film of fluorescent
material, and particularly from the layer of molecules of the material
attached to a wall of the optical waveguide. The propagation region of the
waveguide is defined by a layer of dielectric material. Another wall of
the waveguide is a film of conductive, reflective material on the surface
of the dielectric layer opposite that on which the layer of fluorescent
molecules is deposited. The excitation radiation which is incident on the
fluorescent material is self-coupled to the waveguide to support the
propagation of the modes which generate the strong field. The enhancement
factor of this basic waveguide mode enhancement system can be up to 200.
The addition of corrugations to the basic waveguide enhancement system
provides the directionality, polarization discrimination, increased
enhancement, and emission wavelength selectivity to the emitted
fluorescent light described above.
The directionality of the fluorescence emitted at a given wavelength is
believed to be attributable to the grating induced emission from optical
waveguide modes supported by the structure. The operation of the resulting
directionality may be appreciated by considering an optical waveguide with
a perfectly smooth surface and light of a supported mode propagating
within it. Such light incident on the interior waveguide boundary
evidences total internal reflection and is never detected at points
outside the waveguide. A corrugation pattern imposed on the waveguide
boundary creates a deficiency in the smoothness, allowing the light
incident on the waveguide boundary to be diffracted out of the waveguide.
Thus, the corrugations act in the first order as a diffraction grating on
the incident light. It is well known in the art that light incident on a
diffraction grating demonstrates an intensity distribution with maxima of
intensity at discrete angles with respect to a normal to the mean grating
surface. The discrete angles are a function of the grating dimensions and
the wavelength of the incident light. With a fluorescent sample overlaying
the waveguide boundary, the fluorescence induced is also emitted
directionally. Therefore, for each emitted wavelength there may be a
multiplicity of discrete angular emissions corresponding to the orders of
diffraction.
The polarization discrimination is believed due to the emissions excited by
the various TM and TE modes supported by the waveguide. It is a well known
property of all waveguides that, because of the energy distributions in
each particular mode, each mode of the same wavelength has a different
effective index of refraction in the waveguide medium. Therefore, two
modes of an equivalent wavelength propagating in the waveguide have
different ray-paths or, equivalently, have different incident angles at
the waveguide boundaries. The corrugated waveguide boundary with the
fluorescent layer was analogized to a diffraction grating above. It is
well known that light rays of the same wavelength incident on a
diffraction grating at different angles will have different angles of
diffraction. Since the different TM and TE modes of the same wavelength
have different incident angles on the corrugated surface, they have
different angles of diffraction. With a fluorescent sample over laying the
waveguide boundary, the fluorescent induced is also emitted directionally
as a function of propagation mode.
Additionally, the effective indices of refraction are sensitive to almost
all features of the waveguide, including waveguide thickness, dielectric
layer, 16, dielectric material, conductive layer, 14, conductive material,
etc. Therefore the discrete angles of emission corresponding to each
waveguide propagation mode will shift with changes in these properties.
The increased enhancement of the emitted light is believed due to the
concentration of the emitted light at angles to the normal which are
discrete rather than a diffuse fluorescent emission. The enhancement from
this mechanism acts in conjunction with the enhancement due to the basic
waveguide system discussed above and in U.S. Pat. No. 4,649,280.
It should be noted that the foregoing theoretical discussion is intended to
provide the inventor's present understanding of the phenomena observed in
the invention, and is not to be regarded as a definitive statement on how
the invention functions or to limit the invention in any way.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic diagram of a cross section of a structure which emits
directional, enhanced fluorescence from molecular layers.
FIG. 2 is a schematic diagram illustrating the operation of the invention
and the improved method for stimulating directional, enhanced fluorescence
from molecular layers.
FIG. 3 is a plot illustrating the fluorescence as a function of angle
detected, .phi..sub.d, from the normal to a particular embodiment of the
structure illustrated in FIGS. 1 and 2.
FIG. 4 is a plot showing the enhancement factor as a function of the
thickness of the layer d for particular embodiments of the structures
shown in FIGS. 1 and 2 for a particular wavelength emitted attributed to
the TM.sub.o waveguide mode, where the incident light is TM and TE
polarized.
FIG. 5 is a plot showing the enhancement factor as a function of the
thickness of the layer d for a particular embodiment of the structures
shown in FIGS. 1 and 2 for a particular wavelength emitted attributed to
the TE.sub.o waveguide mode, where the incident light is TM and TE
polarized.
DETAILED DESCRIPTION
Referring to FIG. 1, a substrate, 10, which may be a rectangular glass
slide is coated with a grated layer, 12. The grated layer, 12, in one
configuration, is created by depositing a layer of photoresist on the
glass substrate, imposing an interference pattern on the photoresist, and
then developing the photoresist. Spinning methods commonly employed in
research laboratories and in the semiconductor industry can be used to
achieve a sufficiently uniform thickness of the photoresist on the slide.
In one embodiment the entire slide was coated with a photoresist with a
thickness of approximately 300 nm. The particular thickness is
unimportant, as long as it can support the minimum to maximum requirements
of the corrugation structure, approximately 50 nm peak to valley in one
particular corrugated structure, 12. The area of the interference pattern
and the resulting corrugation pattern does not have to cover the whole
slide. An area of 1 cm.sup.2 was found to be adequate. The photoresist
pattern may be recorded by illuminating the photoresist with the two
recombined beams split off a common laser source. In a particular
embodiment the periodicity of the corrugation, .LAMBDA. in FIG. 1, was
0.636 .mu.m. The developed photoresist produces a shallow corrugated
grating structure, 12. This produces a shallow, corrugated photoresist
underlayer which corrugation appears on later layers impressed upon the
developed photoresist. Other methods of creating shallow gratings, such as
embossing, may be used depending on the particular manufacturing
environment. In a commercial setting relating to medical diagnostics, it
is contemplated manufacturers would use stamped plastic gratings of
predetermined dimensions selected according to the test to be applied to
an unknown sample. Such grating manufacturing technology currently exists.
Periods of corrugation in the range 0.1-1.0 .mu.m are contemplated for
these gratings.
The remainder of the structure which provides the system for directional,
enhanced fluorescence from molecular layers shown in FIG. 1 is created in
a manner analogous to that described in U.S. Pat. No. 4,649,280, the
disclosure of which is incorporated herein by reference thereto. Grating,
12, is coated with conductive layer, 14, of conductive, reflective
material. Vacuum deposition techniques may be used. The thickness of
conductive layer, 14, is on the order of 50 nm, depending on the metal
used. A dielectric layer, 16, of thickness d of a dielectric material is
deposited upon conductive layer, 14. Lithium Fluoride (LiF) among other
dielectric materials may be used. The thickness, d, of dielectric layer,
16, is critical in its relation to the wavelength(s) of the exciting,
incident radiation and the emitted wavelength, as will be apparent from
FIGS. 4 and 5, but is nominally on the order of 20-500 nm. Conductive
layer, 14, and dielectric layer, 16 may be made using high vacuum
thermoevaporation techniques.
Fluorescent material layer, 18, is deposited over dielectric layer, 16. It
is illustrated as the row of spheres to schematically show the molecular
layer at interface, 20, between fluorescent material layer, 18, and
dielectric layer, 16. The fluorescent component in the fluorescent
material layer may be bound to the molecule of interest in accordance with
techniques used in fluorescent assays. The thickness of fluorescent
material layer, 18, is desirably on the order of single molecules in
thickness. This is not an absolute requirement and thickness up to an
optical wavelength, i.e., between 1 and 10 .mu.m, is adequate. It must be
thin enough to allow exciting light to reach molecules near interface 20,
so that waveguide propagation modes are excited through coupling with the
fluorescent molecules absorbing the incident light.
By way of example, fluorescent material layer, 18, may be applied to the
system once conductive layer, 14, and dielectric layer, 16, are applied.
Fluorescent material layer, 18, may be applied using slow heating
evaporation of a fluorescent dye at very low pressure. Alternative methods
are well known in the art and include spin coating and dipping.
Fluorescent material layer, 18, dielectric layer 16, and layer of
reflective conductive material 14 define an optical waveguide which
supports a plurality of propagation modes. Conductive layer, 14, has
corrugated structure analogous to grating, 12, and follows the contours of
grating, 12. Dielectric layer, 16, has corrugated structure analogous to
conductive layer, 14, and follows the contours of conductive layer, 14.
Fluorescent material layer, 18, has corrugated structure analogous to
dielectric layer, 16 it having been deposited on dielectric layer, 16.
It is apparent that the corrugated structure in the film is not limited to
a corrugation pattern in two dimensions as shown in FIG. 1. Grated layer,
12, may have superimposed gratings oriented in a number of directions with
respect to the grating normal, the normal to such corrugated structures in
general defined as the normal to the plane seen from a point an infinite
distance above the corrugated surface. One method of achieving such
overlapping corrugated structure is to deposit a layer of photoresist on a
glass substrate and illuminate the photoresist with two or more
interference patterns oriented at different directions with respect to the
photoresist surface normal prior to developing the photoresist. The
disoriented interference patterns may be achieved using two or more
recombined beams split off two or more common laser sources, each pair of
split beams recombined at the same point on the layer of photoresist, but
the plane defined by each pair of beams distinctly oriented with respect
to the surface normal at the common point of incidence. Similarly, a
plastic grating commercially manufactured could be made with superimposed
non-parallel gratings.
The corrugated surface comprised of a number of such superimposed gratings
has a number of non-parallel "surface profiles", a "surface profile"
defined as the cross-section of the surface on a plane normal to the
corrugated surface oriented so that one of the regular corrugation
patterns is displayed.
Referring to FIG. 2, incident beam, 30, of wavelength .lambda..sub.i passes
through polarizer, 33 and is incident at angle .phi..sub.i with respect to
normal, 22. Both the polarizer 33 and a specific angle of incidence
.phi..sub.i are optional, and serve to enhance the properties of the
present invention, as described below. Due to the interaction of incident
beam, 30, with fluorescent layer 18, one or more fluorescent beams of
wavelength .lambda..sub.f appear, shown in FIG. 2 as fluorescent beam 31,
and fluorescent beam 32. Fluorescent beam 31, is emitted at angle
.phi..sub.d.sbsb.1, with respect to normal, 22, passes through polarizer
34, and is detected by optical detector, 36. Likewise, fluorescent beam,
32, also of wavelength .lambda..sub.f, is emitted at angle
.phi..sub.d.sbsb.2, with respect to normal, 22, passes through polarizer,
35, and is detected by optical detector, 37. Each discretely emitted
fluorescent beam is either TM or TE polarized, and the polarizers, 34, 35,
prior to detection can be rotated to verify this. It is seen that
polarizers 34, 35 are also optional to the present invention, and serve to
only verify the polarization of the emitted wave. The intensity of the
fluorescent beam is a function of the polarization of the incident beam,
30. Therefore, the intensity detected at the optical detectors, 36, 37,
varies as polarizer, 33, is rotated. Each fluorescent beam is attributed
to a particular propagation mode in the waveguide, and the directionality
is attributed to the corrugation structure. As the waveguide thickness, d,
increases, more modes appear, and therefore more discrete fluorescent
beams appear. Furthermore, each mode may interact with more than one order
of the surface corrugation, thereby having more than one discrete emission
angle attributable to one waveguide mode.
In one particular configuration of FIG. 2, the fluorescent material, 18, is
Rhodamine B and the dielectric layer, 16 is LiF. The thickness of the
dielectric layer, d=150 nm and the period of the surface profile,
.LAMBDA.=0.636 micrometer. Incident beam, 30, originates from an Argon ion
laser with wavelength .lambda..sub.i =514.5 nm and power attenuated to 0.1
milliwatt. The angle of incidence .phi..sub.i is adjusted to approximately
15.degree. to maximize the fluorescent output. Optical detectors, 36, 37,
may be used with a monochrometer or other filter to select .lambda..sub.f
=585 nm, and polarizers, 34 or 35, are moved through the angles of
detection with respect to the normal.
Referring to FIG. 3, the sharp peak at approximately -38.degree. shows that
a TM polarized beam of wavelength 585 nm is present at -38.degree.. A
similar peak is also expected at +38.degree., but its actual detection was
prevented due to the experimental apparatus. With the polarizer oriented
in a TE direction, the sharp peak at .+-.5.degree. of wavelength 585 nm
demonstrates a TE polarized beam discretely emitted at that angle. The
fluorescent beam at -38.degree. is attributed to the TM.sub.o waveguide
mode interacting with the 1st order of the grating. The fluorescent beam
at .+-.5.degree. is attributed to the TE.sub.o mode of the waveguide
interacting with the 1st order of the grating.
The diffuse pattern, a, in FIG. 3 is the fluorescent emission as a function
of the detection angle for an uncorrugated waveguide. The wavelength of
the detected light is 585 nm and the diffuse distribution is attributed to
diffuse emission from each waveguide mode. Therefore, unpolarized light is
detected at each point of detection. The radial scale is normalized
separately for the peaked and the diffuse patterns. The diffuse pattern is
for .LAMBDA..fwdarw..infin. in FIG. 1, i.e., a flat surface. The peaks
have an enhancement factor of 200 to 2000 while the enhancement factor of
the diffuse emission does not exceed 200.
FIG. 3 demonstrates that if a known material labeled with a fluorescent
component fluoresces at 585 nm, its use in a system as shown in FIG. 2
will result in strong peaks detected at .+-.5.degree. and .+-.38.degree..
Since the angle of emission is dependent on the interaction of the
particular wavelength with the corrugated structure, the presence of the
material in an unknown sample labeled with a fluorescence is indicated by
fluorescent emission at angles characteristic of the material.
It is further noted that the corrugated waveguide could be manufactured as
described above using an aperiodic grating, resulting in an aperiodic
waveguide surface.
Referring to FIGS. 4 and 5, the enhancement of the excited fluorescence is
shown for a system of the type described in FIG. 2. FIGS. 4 and 5 are
another exemplary case where the angle of incidence of the 514.5 nm
excitation beam is approximately 15.degree.; the fluorescent material is
Rhodamine B; the dielectric layer is LiF; the detector is placed at the
peak detection angle for the particular wavelength detected (585 nm) and
the type of polarization. The intensity detected is then compared with the
fluorescent intensity detected at that angle from the same quantity of
molecules deposited on a glass substrate and similarly excited. The ratio
of the former signal to the latter is the enhancement factor defined above
In FIG. 4, the enhancement factor is plotted as a function of d in FIGS. 1
and 2 for the 585 nm TE wave excited by the TE.sub.o mode for incident
wave TM or TE polarized. In FIG. 5, the enhancement factor is plotted as a
function of d In FIGS. 1 and 2 for the 585 nm TM wave excited by the
TM.sub.o mode for an incident wave TM or TE polarized. From FIG. 4 it is
seen that the enhancement factor for TE-polarized emission can be as high
as 1000 while FIG. 5 shows that the enhancement factor for TM-polarized
emission can be close to 2000.
FIGS. 4 and 5 demonstrate that the dielectric layer thickness ("d" in FIGS.
1 and 2) can be set to maximize the enhancement at a particular angle of
detection (equivalently, from one of the particular waveguide modes).
Alternatively, d may be set to give roughly equal and substantially
increased enhancement at two or more angles of detection (equivalently,
from two or more particular waveguide modes). These figures demonstrate
that when testing an unknown sample for the presence or concentration of a
particular material, the selection of d will substantially improve the
ability to detect the presence or concentration of the material.
The increased detection ability and the unique angular and polarization
features corresponding to a particular wavelength can be combined in a
commercial embodiment which simultaneously performs different analyses on
an unknown sample, leading to more accurate results in less time, with a
minimum of hardware. A configuration like that of FIG. 2 could have one or
more optical detectors fixed at those angles corresponding to the discrete
angular emissions of a particular wavelength excited by propagation modes
of a waveguide of set dimensions and materials. The detection of emission
from an unknown fluorescent material layered on the waveguide at the
various detectors would show the material fluoresced at the known
wavelength. As emission at that wavelength would correspond to a known
material. The system eliminates the need of a monochrometer to test for
the material by determining the wavelength. A polarizer before detection
would further confirm the presence of the particular wavelength
corresponding to the material. Finally, the increased enhancement would
provide the ability to determine concentration of the material, once
reference concentrations and peak limits are determined.
* * * * *
|
|
 |
|
 |
Description |
|
