U.S. patent application number 13/633713 was filed with the patent office on 2013-01-24 for methods for detecting defects in inorganic-coated polymer surfaces.
This patent application is currently assigned to LIFE TECHNOLOGIES CORPORATION. The applicant listed for this patent is Yu-Zhong Zhang. Invention is credited to Yu-Zhong Zhang.
Application Number | 20130020507 13/633713 |
Document ID | / |
Family ID | 47555147 |
Filed Date | 2013-01-24 |
United States Patent
Application |
20130020507 |
Kind Code |
A1 |
Zhang; Yu-Zhong |
January 24, 2013 |
Methods for Detecting Defects in Inorganic-Coated Polymer
Surfaces
Abstract
Lipophilic fluorescent substances can be used to detect surface
defects in materials having hydrophilic (e.g., inorganic) coatings.
Use of the described methods makes surface defects appear
fluorescent, while the remaining surfaces are not labeled. The
disclosed methods are inexpensive, rapid, and easy alternatives to
existing approaches.
Inventors: |
Zhang; Yu-Zhong; (Eugene,
OR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Zhang; Yu-Zhong |
Eugene |
OR |
US |
|
|
Assignee: |
LIFE TECHNOLOGIES
CORPORATION
Carlsbad
CA
|
Family ID: |
47555147 |
Appl. No.: |
13/633713 |
Filed: |
October 2, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12808325 |
Jun 17, 2010 |
8304242 |
|
|
13633713 |
|
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Current U.S.
Class: |
250/459.1 ;
428/141; 548/405 |
Current CPC
Class: |
Y10T 428/24355 20150115;
C07F 5/022 20130101; G01N 21/91 20130101 |
Class at
Publication: |
250/459.1 ;
548/405; 428/141 |
International
Class: |
G01N 21/64 20060101
G01N021/64; B32B 3/30 20060101 B32B003/30; C07F 5/02 20060101
C07F005/02 |
Claims
1. A method of identifying a defect in a surface, comprising: a)
providing a substrate having a hydrophobic surface at least
partially coated by a hydrophilic layer, wherein the hydrophilic
layer has the defect therein; b) contacting the substrate with a
lipophilic, fluorescent substance, for a sufficient amount of time
for the substance to contact the defect; c) exciting the
fluorescent substance with energy at an appropriate wavelength to
generate a detectable fluorescence response; and, d) detecting the
fluorescence response of the substance.
2. The method of claim 1, further comprising washing the substrate
after contacting the substrate with the lipophilic, fluorescent
substance.
3. The method of claim 1 wherein the substrate comprises a
polymer.
4. The method of claim 1 wherein the hydrophilic layer is or
comprises an inorganic material.
5. The method of claim 1 wherein the lipophilic, fluorescent
substance is a fluorescent compound that comprises a
4,4-difluoro-4-bora-3a,4a-diaza-s-indacene moiety.
6. The method of claim 1 wherein the lipophilic, fluorescent
substance further comprises a lipophilic moiety.
7. The method of claim 6 wherein the lipophilic, fluorescent
substance further comprises two or more lipophilic moieties.
8. The method of claim 6 wherein the lipophilic moiety is a
hydrocarbon having 1-20 carbon atoms.
9. The method of claim 6 wherein the lipophilic moiety is an alkyl
group having 1-20 carbon atoms.
10. The method of claim 6 wherein the lipophilic moiety is phenyl
or styryl group.
11. The method of claim 1 wherein the hydrophilic layer is less
than 10 .ANG. in thickness.
12. The method of claim 1 wherein the lipophilic, fluorescent
substance is associated with a microparticle.
13. The method of claim 1 wherein the lipophilic, fluorescent
substance is a semiconductor nanocrystal.
14. A lipophilic, fluorescent substance that comprises a
4,4-difluoro-4-bora-3a,4a-diaza-s-indacene moiety and a lipophilic
moiety.
15. The lipophilic, fluorescent substance of claim 14 wherein the
lipophilic moiety is a hydrocarbon having 1-20 carbon atoms.
16. The lipophilic, fluorescent substance of claim 14 wherein the
lipophilic moiety is an alkyl group having 1-20 carbon atoms.
17. The lipophilic, fluorescent substance of claim 14 wherein the
lipophilic moiety is a phenyl or styryl group.
18. The lipophilic, fluorescent substance of claim 14 wherein the
substance is associated with a microparticle.
19. A substrate comprising an identifiable defect: a) a hydrophobic
surface, a substrate having a hydrophobic surface at least
partially coated by a hydrophilic layer, wherein the hydrophilic
layer has the defect therein; and b. a lipophilic, fluorescent
substance that is in contact with the defect.
20. The substrate of claim 19 wherein the lipophilic, fluorescent
substance comprises a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene
moiety and a lipophilic moiety.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 61/014,396, filed Dec. 17, 2007, the disclosure of
which is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The invention relates to methods for detecting surface
defects in coated substrates (e.g., inorganic-coated polymer
surfaces) and, more specifically, to the use of hydrophobic
fluorescent materials to detect surface defects.
DESCRIPTION OF RELATED ART
[0003] Polymers are commonly used materials because of their
flexibility, light weight, and low cost. Many polymer properties
could be enhanced by the addition of an inorganic coating on their
surface. This inorganic film could serve as a gas diffusion barrier
for various packaging applications. The inorganic layer could also
serve to protect the underlying polymer and give the polymer higher
strength. Unfortunately, inorganic layers are difficult to deposit
on polymers because the deposition of inorganic materials is
usually performed at temperatures above the melting temperature of
the polymer.
[0004] Alumina-coated polymer surfaces are widely used in
industrial and consumer products. Atomic layer deposition ("ALD",
sometimes alternatively referred to as chemical vapor deposition
"CLD") is used to deposit a thin nanometer layer of alumina or
other inorganic or metallic materials onto a polymer surface. ALD
coatings can be used to insulate, facilitate charge dissipation and
functionalize the surface of MEMS (Micro-Electro-Mechanical
Systems) devices. Polyimides are commonly used as the polymer.
Materials such as alumina-coated polyimides are used in the
semiconductor industry to prepare high vacuum, vapor resistant
sealing packages for semiconductor wafers.
[0005] Due to inherent limitations and variability in atomic layer
deposition, various defects such as non-coated areas, surface
irregularities, cracks, or scratches can be introduced during large
scale production. These defects can be introduced during the
deposition of the inorganic layer, or afterwards during further
processing or handling.
[0006] Several quality control testing procedures exist, but tend
to be costly, time consuming, and expensive. One example is a
helium leakage test. This test measures the vapor transmission rate
through the polymer as an indicator of the integrity of the
inorganic coating. This test can indicate the presence of a defect,
but does not allow detection of the exact location of the
defect.
[0007] Other tests for water vapor permeation include gravimetric
(loss of water or gain of water on P.sub.2O.sub.5), capacitive or
resistive (using a humidity sensor), spectroscopy, calcium
degradation (either optical or change in resistance), and
radioactive (using tritium or .sup.14CO).
[0008] A publication described a method for detecting under-film
corrosion using a hand-held UV lamp (D. E. Bryant and D.
Greenfield, Progress in Organic Coatings, 57(4): 416-420 (2006)).
The chemical 8-hydroxyquinoline-5-sulfonic acid hydrate was used to
study corrosion of coated aluminum, and 9-anthyl-5-(2-nitrobenzoic
acid) disulfide was used with iron. Metals coated with various
polymers were scored with a scalpel, allowing corrosion to
occur.
[0009] Atomic force microscopy ("AFM") was used to examine surface
cracks on an indium tin oxide coated polycarbonate substrate that
had been subjected to cycles of bending (L Ke et al., Applied
Physics A: Materials Science & Processing, 81(5): 969-974
(2005)). AFM showed that bending increases the roughness of the
inorganic coating surface. Calcium degradation test showed that
surface cracks are perpendicular to the flexing direction, and that
barrier performance deteriorated after bending. An organic light
emitting device ("OLED" or "organic LED") fabricated on the surface
showed decreased electrical and optical performance due to moisture
and oxygen permeation.
[0010] An auto-optical inspection system ("AOI") for detection of
defects in OLEDs was described by D. B. Perng et al., Journal of
Physics: Conference Series, 13: 353-356 (2005). The publication
indicated that OLED defects commonly include dark points,
non-uniform luminescence, surface scratches, insufficient rubber
widths, and lack of color uniformity. The computer-controlled AOI
is based on a lighting mechanism including a conducting fixture, a
UV light, a coaxial LED light, and a back light.
[0011] Various U.S. patents have issued offering methods for
detecting surface defects of materials.
[0012] U.S. Pat. No. 4,968,892 (issued Nov. 6, 1990) describes a
testing apparatus for identifying surface defects in a workpiece.
The piece is treated with a fluorescent substance that is trapped
in flaws in the surface. The apparatus includes a light source,
lenses, and filter to scan the surface.
[0013] U.S. Pat. No. 5,723,976 (issued Mar. 3, 1998) describes a
method for detecting defects in encapsulated electronic components.
The method involves immersing the component in an aqueous
florescent solution of a water-soluble fluorescent substance that
fluoresces when moistened, but that does not fluoresce when dry.
The component is visualized in humid air, and then in dry air to
detect fluorescence (when moist) and lack of fluorescence (when
dry) at a defect.
[0014] U.S. Pat. No. 5,965,446 (issued Oct. 12, 1999) suggests a
method of detecting defects in surfaces. A solution of fluorescent
molecules in a volatile organic solvent is prepared, and applied
across the surface using a slip of paper. The paper is used to
uniformly distribute the solution across the surface before the
organic solvent evaporates.
[0015] U.S. Pat. No. 6,097,784 (issued Aug. 1, 2000) offers a
method for amplifying defects connected to a top surface of a
semiconductor device. A dye is applied to the top surface, and
leeched into a developing gel. The gel develops defect indications
that are more easily visualized than the defects themselves. The
dye can be a fluorescent dye.
[0016] U.S. Pat. No. 6,427,544 (issued Aug. 6, 2002) suggests an
environmentally friendly method for detecting defects in parts. The
parts are submerged in a mixture of a penetrant dye and
supercritical carbon dioxide. The part is removed, and inspected
for the presence of dye in any defects. The dye can be a
fluorescing penetrant dye that is visualized with UV light.
[0017] U.S. Pat. No. 6,677,584 (issued Jan. 14, 2004) offers a
manufacturing fluid containing a fluorescent dye. A component is
either ground or cut in the presence of the manufacturing fluid,
and the component is subsequently inspected for surface cracks or
defects. The manufacturing fluid can be especially useful in
processing of ceramic parts.
[0018] U.S. Pat. No. 6,916,221 (issued Jul. 12, 2005) describes an
optical method for determining defects in OLEDs. A digital image of
the excited OLED surface is obtained, and a computer or user
inspects the image to determine defects.
[0019] U.S. Pat. No. 6,943,902 (issued Sep. 13, 2005) describes a
method of determining layer thickness or respective amount of
filling, layer thickness distribution, defect, accumulation or
inhomogeneity within a material layer. The material is mixed with
an agent that absorbs radiation before the layer is prepared. The
layer is irradiated, and the emitted light is detected. In this
method, the agent is permanently embedded throughout the layer.
[0020] While inorganic-coated polymers are widely used in industry,
surface defects degrade, and potentially eliminate, the desirable
properties of the materials. For example, a defect may allow water
to penetrate the material, or may reduce the ability of the
material to hold a vacuum. Thus, despite efforts made to date,
simple, reliable methods to verify the integrity of materials, or
conversely, simple, reliable methods to detect surface defects of
materials are still needed. Additionally, methods that provide
detection of the location of the defects are desirable.
SUMMARY OF THE INVENTION
[0021] Surface defects in materials having a polymer layer coated
with a hydrophilic layer (e.g., a surface layer of an inorganic
substance) can be detected and localized using at least one
lipophilic fluorescent substance. Contacting the material with the
fluorescent substance renders any surface defects fluorescent,
while the remaining surface lacking defects is not labeled.
[0022] In one aspect, a method is provided for identifying a defect
in a surface. The method involves a) providing a substrate having a
hydrophobic surface at least partially coated by a hydrophilic
layer, wherein the hydrophilic layer has the defect therein; b)
contacting the substrate with a lipophilic, fluorescent substance,
for a sufficient amount of time for the substance to contact the
defect; c) exciting the fluorescent substance with energy at an
appropriate wavelength to generate a detectable fluorescence
response; and d) detecting the fluorescence response of the
substance. The method can further include washing the substrate
after contacting the substrate with the lipophilic, fluorescent
substance. The substrate can include a polymer. The hydrophilic
layer can be or include an inorganic material (e.g., a metal
oxide). The hydrophilic layer is typically less than 10 .ANG. in
thickness. The lipophilic, fluorescent substance can be a
fluorescent compound that includes a
4,4-difluoro-4-bora-3a,4a-diaza-s-indacene moiety. The lipophilic,
fluorescent substance can further include a lipophilic moiety or
can include two or more lipophilic moieties. For example, the
lipophilic moiety can be a hydrocarbon having 1-20 carbon atoms,
such as an alkyl group having 1-20 carbon atoms or a phenyl or
styryl group. The lipophilic, fluorescent substance can be
associated with a microparticle or a semiconductor nanocrystal.
[0023] In another aspect, a lipophilic, fluorescent substance is
provided that includes a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene
moiety and a lipophilic moiety. The lipophilic moiety can be a
hydrocarbon having 1-20 carbon atoms, such as an alkyl group having
1-20 carbon atoms or a phenyl or styryl group. The lipophilic,
fluorescent substance can be associated with a microparticle.
[0024] In yet another aspect, a substrate is provided that includes
an identifiable defect. The substrate can include a hydrophobic
surface that is at least partially coated by a hydrophilic layer
(e.g., an inorganic material) in which there is a defect. A
lipophilic, fluorescent substance can be in contact with the
defect. Any lipophilic, fluorescent substance can be in contact
with the defect, including, for example, a substance that includes
a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene moiety and a
lipophilic moiety.
[0025] In yet another aspect, kits are provided including a
lipophilic, fluorescent substance and, optionally, additional
components for carrying out the disclosed methods.
[0026] The compositions, kits, and methods provided herein offer
numerous advantageous over traditional approaches for visualizing
surface defects and provide an inexpensive, rapid, and relatively
easy to use alternative to existing approaches.
BRIEF DESCRIPTION OF THE FIGURES
[0027] FIG. 1 is an image demonstrating that lipophilic,
fluorescent molecules adhere selectively to scratches introduced in
an ALD alumina coating.
[0028] FIG. 2 shows images of cracks in an ALD alumina coating
rendered visible by a lipophilic, fluorescent substance: (A) series
of channel cracks generated at the specimen's interior after a 5%
externally applied strain, (B) cracks at edge of specimen resulting
from shearing during sample preparation, (C) FESEM image
demonstrating the true size of a single shear crack.
[0029] FIG. 3 shows images of individual defects in/on an
Al.sub.2O.sub.3 ALD film rendered visible by a lipophilic
fluorescent tag: (A) defect density and location revealed relative
to a marker at low magnification via confocal microscopy; (B, C)
details of size and morphology of defects at site #1 and site #2 in
(A) identified by high magnification FESEM.
DETAILED DESCRIPTION OF THE INVENTION
[0030] Before describing the present invention in detail, it is to
be understood that this invention is not limited to specific
compositions or process steps. It should be noted that, as used in
this specification and the appended claims, the singular form "a",
"an" and "the" include plural references unless the context clearly
dictates otherwise. It also should be noted that the term "about",
when used to describe a numerical value, shall encompass a range up
to .+-.15% of that numerical value, unless the context clearly
dictates otherwise. While compositions and methods are described in
terms of "comprising" various components or steps (interpreted as
meaning "including, but not limited to"), the compositions and
methods can also "consist essentially of" or "consist of" the
various components and steps, such terminology should be
interpreted as defining essentially closed-member groups.
[0031] Compositions and Methods of Use
[0032] Methods are provided for the detection of defects in a
material. One embodiment of the invention is directed towards
methods for the detection of surface defects in a material. The
material can comprise, for example, a polymer layer coated with an
inorganic layer. The method can comprise contacting the material
with at least one lipophilic fluorescent substance to allow the
substance to localize at the defect, and detecting the localized
substance. The method can be used to detect various types of
surface defects, including, without limitation, mechanical defects,
such as cracks, pin holes, surface irregularities, scratches,
uncoated areas, flaking, or any other type of defect that can arise
during production or handling of the material. Also provided are
methods for detecting the presence of surface particles or
contaminants (e.g., grease, oil, dust, fibers, and the like). The
present methods can be used for detecting defects that traverse the
full thickness of a surface layer on a substrate. For example, a
crack or pin hole in a hydrophilic coating that leaves a portion of
an underlying hydrophobic substrate exposed can be detected using
the subject lipophilic fluorescent substances. The disclosed
fluorescent substances can label surface defects ranging in size
from several to hundreds of nanometers to microns or larger. For
example, defects can be identified having a width or diameter
(e.g., in the case of a pinhole defect) of about 500 nm or less; or
about 250 nm or less; or about 100 nm or less; or 50 nm or
less.
[0033] The material can generally be any material. Examples of
materials include OLEDs (organic light-emitting diodes), thermal
ground planes, solar panels, films and bags (such as those used the
electronics, food packaging, or medical device industries), fiber
optics, flexible displays, liquid crystal display (LCD) assemblies,
flat panel displays, magnetic information storage media (MIS),
Micro-Electro-Mechanical System (MEMS), and Ultra large-scale
integration (ULSI) circuits.
[0034] The material can include a polymer or a combination of
polymers. The polymer can be in the form of a surface layer. The
polymer or polymer layer can generally be any polymer. The polymer
is preferably hydrophobic. Examples of polymers include
polystyrene, polyurethane, polyimide, epoxy, polyethersulfon (PES),
polyethylene naphthalate (PEN), HSPEN (heat stabilized polyethylene
naphthalate), KAPTON (a polyimide membrane commercially available
from DuPont), polyetheretherketone (PEEK), polysulfone (PSF),
polyetherimide, polyethylene phthalate, and polyethylene
terephthalate (PET). The polymer can generally be in any
three-dimensional configuration. Examples of configurations include
planar sheets, films, coatings, tubing, fibers, and beads.
[0035] The material (e.g., a substrate formed of a polymer) can
further include at least one layer of a coating. The coating can be
a continuous coating (e.g., a film) or can cover only a portion of
the substrate. The coating can include any type of material (e.g.,
a polymer or an inorganic material), so long as it is less
hydrophobic than the substrate material. For example, the coating
can be a hydrophilic material (e.g., a hydrophilic polymer or an
inorganic material). In certain embodiments, the coating is an
inorganic layer. The inorganic layer can generally be any inorganic
layer. The inorganic layer is preferably hydrophilic. Inorganic
layers can be metal oxide layers. Inorganic layers can be
metal-anion solids such as ZnS, GaP, Ta.sub.2O.sub.3,
Al.sub.2O.sub.3 (alumina), TiO.sub.2, GeO.sub.2, and VO.sub.x. The
coating can range in thickness. For example, the coatings can be
less than about 1 micron; or less than about 500 nm; or less than
about 100 nm; or less than about 50 nm. The coating can be applied
or deposited onto a substrate by any means known used in the art.
In certain embodiments, an inorganic coating layer is applied by
atomic layer deposition (ALD) to form a film on the substrate
having a thickness of 50 nm or less (e.g., about 25 nm).
[0036] The lipophilic fluorescent substance can generally be any
lipophilic fluorescent substance. Examples of lipophilic
fluorescent substances include fluorescent dyes, fluorescent
microspheres, and quantum dots (sometimes referred to as
semiconductor nanocrystals). The fluorescent substance can generate
fluorescence prior to application to the sample or can generate
fluorescence during use (e.g., upon contact with the sample). For
example, lipophilic, fluorescent substances can appear
non-fluorescent or minimally fluorescent when dissolved in an
aqueous solution, such as water or a buffer. However, when in a
hydrophobic environment (e.g., when in contact with a hydrophobic
surface), certain lipophilic fluorescent substances (e.g.,
diaza-indacenes, squarenes and some styryl dyes) can produce an
intense fluorescence signal. The type of lipophilic fluorescent
substance used in the present methods can vary depending on, for
example, the composition and configuration of substrate and
coating, the thickness of the coating, and the type and size of
defect. Lipophilic fluorescent substances are generally of a size
that permits ready entry into nanometer-scale defects. Certain
substances provided herein are relatively small molecules (e.g.,
molecular weight of about 200-400). However, larger fluorescent
substances with dimensions in the nanometer or micron range may be
desirable for detecting larger defects. Lipophilic fluorescent
substances are typically hydrophobic compounds or substances that
tend to be non-polar and are not considered water soluble.
Liphophilic substances tend to dissolve in non-polar solvents, such
as, methylene chloride, isopropanol, ethanol, hexane, and the like,
and have no affinity or a negligible affinity for hydrophilic
surfaces. The fluorescent substance itself may be lipophilic.
Alternatively, the fluorescent substance includes a fluorescent
moiety and a lipophilic moiety (e.g., a lipophilic pendant group).
Certain fluorescent moieties can be lipophilic. In certain
embodiments, the fluorescent moiety is relatively less lipophilic
than the lipophilic moiety. Certain fluorescent moieties can be
hydrophilic. A fluorescent substance having a fluorescent moiety
that is relatively less hydrophobic than the pendant moiety can be
used when it is desired that the substance orient itself relative
to the surface. For example, when deposited onto a hydrophobic
surface, a lipophilic pendant group can adhere to the surface
(e.g., by hydrophobic interactions), while the relatively less
hydrophobic fluorescent moiety can present itself away from the
surface. The lipophilic moiety can be bonded (e.g., covalently
bonded) to a fluorescent molecule and can further include a spacer
that can distance the lipophilic moiety from the fluorescent
moiety. In certain embodiments, the fluorescent molecule can
include more than one pendant group, where the pendant groups can
be the same or different. For example, the compound can include 2
or 3 or 4 or more lipophilic groups, which can be the same or
different. Lipophilic groups are typically chosen so as not to
interfere with the fluorescence properties of the fluorescent
molecule. Any type of lipophilic or hydrophobic group can be used
in the preparation of lipophilic, fluorescent substances and are
well known to those skilled in the art. A representative class of
lipophilic moieties includes hydrocarbons. Hydrocarbons can be
saturated or unsaturated, linear, branched, or cyclic, and can
include aliphatic, and/or aromatic moieties. Certain hydrocarbons
include a conjugated hydrocarbon chain. Exemplary lipophilic
hydrocarbon moieties include alkyl groups having 1-20 carbon atoms.
For example, the lipophilic moiety can be a saturated alkyl group
having 10 or less carbon atoms (e.g., 1 to 3; or 3 to 5; or 5 to 7;
or 7 to 9; or 10), which can be substituted or unsubstituted (e.g.,
methyl, ethyl, propyl, butyl, and the like). Alternatively, the
lipophilic moiety can be an unsaturated hydrocarbon, which can be
substituted or unsubstituted, or a conjugated hydrocarbon with
alternating single and double bonds. Other exemplary lipophilic
moieties are or include an aromatic moiety, such as phenyl or
styryl. Other classes of lipophilic moieties include compounds that
contain a heteroatom, such as N, S, O, or a halogen. Yet other
classes of lipophilic substances include fatty acids, fatty
sulfonic acids or fatty sulfates (such as sodium dodecyl sulfate).
In certain embodiments, the fluorescent substance is substituted
with 2 to 4 or more lipophilic pendant groups, which can be the
same or different. For example a fluorescent substance can be
substituted with 2 or 3 or 4 alkyl groups, where each alkyl group
has 1-20 carbon atoms (e.g., methyl, ethyl, propyl, butyl, and the
like). In other embodiments, the fluorescent substance is
substituted with more than one type of lipophilic group. For
example, a fluorescent substance can be substituted with a
combination of moieties, such as, for example hydrocarbon moieties
(e.g., linear or branched alkyl, phenyl, styryl, or the like).
[0037] Examples of lipophilic fluorescent substances include
fluorescent dyes, fluorescent microspheres or microparticles, and
quantum dots (sometimes referred to as semiconductor nanocrystals).
Any type of fluorescent dye may be used in the practice of the
described methods. In certain embodiments, the fluorescent dye can
absorb or emit radiation in the visible range of the
electromagnetic spectrum. Alternatively, the fluorescent dye can
absorb or emit radiation in the near IR region of the spectrum.
Near IR dyes can be effectively used to visualize defects in
substrates that generate background fluorescence. Many fluorescent
compounds tend to lose fluorescence emission intensity (referred to
as "photobleaching") upon sustained illumination (e.g., from
seconds to longer exposure times). Photobleaching arises for
various reasons, including, for example, irreversible modification
of the dye structure. Lipophilic, fluorescent substances are
provided that resist photobleaching and are, therefore, well-suited
for use in detection and characterization of defects according to
the methods provided herein.
[0038] Loss of fluorescence emission intensity also can occur when
a fluorescent substance is present in a high concentration or
agglomerates. Thus, it is generally desired to use a lipophilic
fluorescent compound in the practice of the disclosed methods that
exhibits minimal or no loss in fluorescence emission intensity when
present at a high concentration (e.g., when localized in or on a
defect), or exhibits a loss in intensity or at a rate that is
slower than the time scale of the detection period (such that the
loss in intensity does not affect the measurement). Provided herein
are fluorescent substances that are sufficiently lipophilic to
adhere to hydrophobic substrates (e.g., polymers) and yet maintain
their fluorescence emission intensity when deposited in or on a
surface defect. In contrast to many fluorescent compounds, it has
been found that certain lipophilic, fluorescent compounds provided
herein (e.g., BODIPY dyes) actually exhibit an increase in
fluorescence signal intensity when present in high concentration.
Particular dyes, wherein the dye is hydrophobic, provided herein
(e.g., BODIPY) maintain or increase fluorescence signal intensity
when used to detect surface defects. This unique attribute is
particularly advantageous in the imaging of micron or nanometer
sized defects containing minute quantities of the fluorescent
material. Although not necessary for the practice of the described
methods, particular lipophilic, fluorescent compounds provided
herein also can exhibit a shift in emission wavelength (e.g.,
red-shift) when localized within a defect. Compounds that exhibit a
spectral shift towards longer wavelengths can be used, for example,
to visualize defects in substrates that produce background
fluorescence.
[0039] One representative class of hydrophobic fluorescent dyes
that is suitable for detecting surface defects includes compounds
having a boron dipyrromethene (abbreviated as BODIPY) core
structure (e.g., compounds having a
4,4-difluoro-4-bora-3a,4a-diaza-s-indacene core). BODIPY-based
compounds can be substituted with one or more lipophilic pendant
groups, as described herein. Particular examples of BODIPY-based
fluorescent compounds that can be utilized in the described methods
include those substituted with, for example, a hydrocarbon, such as
methyl, propyl, phenyl, or styryl. For example, representative
BODIPY compounds include, 1,3,5,7,8-pentamethyl BODIPY and
1,3-di-n-propyl BODIPY.
[0040] Other examples of BODIPY-based fluorescent compounds that
can be utilized in the described methods include
4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene;
4,4-difluoro-1,3-dimethyl-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene;
4,4-difluoro-1,3,5,7-tetraphenyl-4-bora-3a,4a,8-triaza-s-indacene;
4,4-difluoro-1,3-diphenyl-5-(2-pyrrolyl)-4-bora-3
a,4a-diaza-s-indacene;
4,4-difluoro-1,3-dipropyl-4-bora-3a,4a-diaza-s-indacene;
4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3 a,4a-diaza-s-indacene,
4,4-difluoro-1,3-diphenyl-5,7-dipropyl-4-bora-3a,4a-diaza-s-indacene;
4,4-difluoro-1-phenyl-3-(4-methoxyphenyl)-5-(2-pyrrolyl)-4-bora-3a,4a-dia-
za-s-indacene;
difluoro(1-((3-(4-methoxyphenyl)-2H-isoindol-1-yl)methylene)-3-(4-methoxy-
phenyl)-1H-isoindolato-N.sup.1,N.sup.2)boron;
difluoro(5-methoxy-1-((5-methoxy-3-(4-methoxyphenyl)-2H-isoindol-1-yl)met-
hylene)-3-(4-methoxyphenyl)-1H-isoindolato-N.sup.1,N.sup.2)boron;
4,4-difluoro-2-ethyl-1,3,5,7,8-pentamethyl-4-bora-3
a,4a-diaza-s-indacene;
4,4-difluoro-1,3-dimethyl-5-styryl-4-bora-3a,4a-diaza-s-indacene;
4,4-difluoro-3,5-di(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-indacene;
3-decyl-4,4-difluoro-5-styryl-4-bora-3 a,4a-diaza-s-indacene;
4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-indace-
ne; 4,4-difluoro-1,3-dimethyl-5-(2-thienyl)-4-bora-3
a,4a-diaza-s-indacene;
difluoro(1-((3-(2-(5-hexyl)thienyl)-2H-isoindol-1-yl)methylene)-3-(2-(5-h-
exyl)thienyl)-1H-isoindolato-N.sup.1,N.sup.2)boron;
4,4-difluoro-1,3,5,7-tetraphenyl-4-bora-3 a,4a-diaza-s-indacene;
4,4-difluoro-1,3-dimethyl-5-(2-(5-methoxycarbonyl-4-methyl-2-oxazolyl)eth-
en yl)-4-bora-3 a,4a-diaza-sindacene; and
difluoro(5-methoxy-1-((5-methoxy-3-(2-(5-(4-methoxyphenyl))thienyl)-2H-is-
oindol-1-yl)methylene)-3
(24544-methoxyphenyl))thienyl)-1H-isoindolato-N.sup.1,N.sup.2)boron.
[0041] Yet other examples of hydrophobic fluorescent dyes that can
be used in the practice of the described methods include
p-aminophenyl phosphorylcholine, naphthalenes, anthracenes,
phenanthrenes, indoles, carbazoles, stilbenes, benzimidazoles,
benzoxazoles, benzothiazoles, quinolines, benzoxanthrones,
oxazoles, isoxazoles, oxadiazoles, benzofurans, pyrenes, perylenes,
coronenes, coumarins, carbostyryls, bimanes, acridines,
polyphenylenes such as terphenyl, alkenyl and polyalkenyl dyes
(including 1,6-diphenyl-1,3,5-hexatriene and
1,1,4,4-tetraphenyl-1,3-butadiene).
[0042] Other long wavelength dyes such as luminescent phenoxazones,
oxazines and pyronines (including nile red); porphines, porphyrins,
phthallocyanines and their metallated complexes, including
complexes with rare earth ions such Eu.sup.3+ and Tb.sup.3+;
xanthenes (including fluoresceins and rhodamines); cyanine,
carbocyanines and merocyanines (including styryl dyes; hydrocarbon
derivatives such as rubrenes and azulenes; are suitable provided
that they are either electrically neutral; or their ionic charges
are balanced by lipophilic counterions that include but are not
limited to lipophilic ammonium salts (such as
hexadecyltrimethylammonium or benzyltrimethylammonium), fatty
acids, fatty sulfonic acids or fatty sulfates (such as sodium
dodecyl sulfate), detergents such as anionic or cationic
derivatives of cholic acids, tetraarylphosphonium or
tetraarylboride; or they contain a suitable functional group (as
described above) for copolymerization.
[0043] The fluorescent lipophilic substance may be contained within
or on a microparticle (e.g., a spherical particle). Microparticles
include those sized so as to be able to readily enter a defect
(e.g., a crack or pinhole) in a surface. In certain embodiments,
the fluorescent lipophilic substance is contained within or on a
microparticle having low surface charge (e.g., particles with
lipophilic surfaces). Microparticles can generally have any shape
or size. For example, microparticles can be sized to have a
dimension along the longest axis that can be about 5 nm to about 20
.mu.m. Certain microparticles can be spherical (referred to as
microspheres). The spherical microspheres can generally have any
diameter. Generally, the diameter (or length across the longest
dimension, in the case of non-spherical particles) can be about 5
nm to about 20 .mu.m. Presently preferred diameters are about 10
.mu.m to about 100 nm. Specific examples of diameters include about
5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, about 10 nm,
about 11 nm, about 12 nm, about 13 nm, about 14 nm, about 16 nm,
about 17 nm, about 18 nm, about 19 nm, about 20 nm, about 21 nm,
about 22 nm, about 23 nm, about 24 nm, about 25 nm, about 26 nm,
about 27 nm, about 28 nm, about 29 nm, about 30 nm, about 40 nm,
about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm,
about 100 nm, about 200 nm, about 300 nm, about 400 nm, about 500
nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, about 1
.mu.m, about 2 .mu.m, about 3 .mu.m, about 4 .mu.m, about 5 .mu.m,
about 6 .mu.m, about 10 .mu.m, about 20 .mu.m, about 30 .mu.m,
about 1 .mu.m, about 2 .mu.m, about 3 .mu.m, about 4 .mu.m, about
10 .mu.m, about 20 .mu.m and ranges between any two of these
values. In certain embodiments, particles of less than 1 micron; or
less than 500 nm; or less than 100 nm are suitable for use in
methods to visualize micron to nanometer-sized defects.
[0044] The spherical microspheres can be prepared from generally
any material. It is presently preferred that the spherical
standards are prepared from a polymer material. Example polymer
materials include polymers and copolymers of styrenes and divinyl
benzenes; an acrylate or methacrylate ester; an acrylic acid or
methacrylic acid; an acrylamide or methacrylamino; an acrylonitrile
or methacrylonitrile; vinyl and vinylidene halides, esters and
ethers; alkenes, including ethylene, propylene, butadiene and
isoprene; epoxides and urethanes.
[0045] The spherical microspheres can be stained with at least one
fluorescent compound, such as the dyes described above. The one or
more fluorescent dyes in the spherical microspheres can be selected
to match the excitation source (e.g., a laser such as an argon-ion
laser, a krypton-argon laser, or a helium-neon laser, a LED, UV
lamp, etc.), and/or an optical filter commonly used in scanner,
fluorescence microscopes or confocal laser-scanning microscopes.
For example, certain microspheres for use according to the
described methods have diameters of about 10 nm or less and low
surface charge loaded with a fluorescent dye that emits
fluorescence in the green region of the electromagnetic
spectrum.
[0046] The lipophilic, fluorescent substance can be a quantum dot
(also referred to as a "semiconductor nanocrystal" or
"nanocrystal"). Quantum dots are nanometer-scale atom clusters
formed of a core (typically zinc sulfide), a semiconductor shell,
and coating. A lipophilic moiety can be attached to the polymer
coating or directly to the semiconductor shell to create
nanocrystals that meet specific defect detection requirements and
to minimize non-specific attachment of the quantum dot to the
surface. The surface of the quantum dots can be relatively
hydrophobic, and can interact with un-coated (defect of ALD
deposition) polymer (e.g., membrane) surfaces by hydrophobic
interactions and lead to defect detection. Typically, quantum dots
can be excited with shorter wavelength excitation sources such as
UV or violet laser and can emit at wavelengths in the visible
region to the near infrared region of the electromagnetic spectrum
(e.g., at about 500 to about 800 nm). Quantum dots can produce a
bright fluorescence signal and are typically more photostable and
less susceptible to bleaching than traditional organic
fluorophores, even under high power excitation over extended
periods of time (e.g., up to several hours). Further, quantum dots
do not generally exhibit a marked reduction in fluorescence
emission intensity when accumulated at higher concentration. As was
discussed with reference to certain classes of lipophilic
fluorescent dyes, quantum dots that maintain fluorescence emission
intensity during use are generally desirable for use in
visualization of surface defects. Quantum dots can generally be any
color quantum dot. Examples of currently commercially available
quantum dots suitable for use in the disclosed methods include the
QDOT nanocrystal products, such as QDOT 525 nanocrystals, QDOT 545
nanocrystals, QDOT 565 nanocrystals, QDOT 585 nanocrystals, QDOT
605 nanocrystals, QDOT 625 nanocrystals, QDOT 655 nanocrystals,
QDOT 705 nanocrystals, and QDOT 800 nanocrystals, all available
from Invitrogen Corporation (Carlsbad, Calif.).
[0047] The lipophilic fluorescent substance can be directly applied
to the material to be analyzed. Alternatively, the fluorescent
substance can be present in a liquid solution or suspension. For
example, the liquid or suspension can comprise water and an organic
solvent, such as DMSO, DMF, toluene, alcohol (such as methanol,
ethanol, or 2-propanol), methylene chloride, and the like, or
mixtures thereof.
[0048] Methods are provided for identifying a defect in or on
coated substrate using a lipophilic fluorescent substance. A
surface of the substrate can be coated in its entirety, or only a
portion of the surface can be coated with a coating. In one method,
a hydrophobic polymer substrate coated with a hydrophilic coating,
such as alumina, is treated with a solution of a lipophilic
fluorescent substance. The fluorescent molecule or material can
selectively bind to defect sites, based on the surface adhesion
characteristics of the system. The contacting step can generally
comprise any suitable application method, such as dipping the
material into a liquid solution or suspension, spraying a liquid
solution or suspension onto the material, spraying the lipophilic
fluorescent substance directly onto the material, rolling the
fluorescent substance directly onto the material, or combinations
thereof. The lipophilic substance can contact the defect (e.g.,
crack) and adhere to the underlying polymer substrate left exposed
by the defect. Lipophilic substances are provided that can easily
enter into nanometer-scale defects, such as typically found, for
example, in ALD barrier coatings. The lipophilic substance can
adhere to the hydrophobic substrate via any type of non-covalent
interaction (e.g., hydrophobic bonding) by virtue of its
hydrophobicity. Further, the lipophilic substance has little or no
affinity for the hydrophilic surface coating and can be readily
removed from the hydrophilic surface.
[0049] The method can further comprise washing the material after
the contacting step and before the detecting step to remove any
non-localized substance. The washing step can be performed once, or
multiple times.
[0050] The detecting step can qualitatively detect the presence or
absence of localized lipophilic fluorescent substance, or can
quantitatively detect the amount of localized lipophilic
fluorescent substance. The detecting step can also determine the
location of the localized lipophilic fluorescent substance on the
material. The detecting step can comprise applying radiation to the
material in order to detect fluorescence emitted from the localized
lipophilic fluorescent substance. The particular type and
wavelength of radiation is selected based upon the spectral
characteristics of the fluorescent substance, and is well within
the talents of the skilled artisan. Examples and sources of
radiation include UV light, lasers, LED light, and visible light.
The detecting step can further comprise preparing an image of the
material. The image can be a film image or an electronic image.
Depending on the size of the defect, a microscope may be useful in
detecting and preparing an image of the defect. The fluorescence
emission of the localized lipophilic fluorescent substance allows
direct identification and localization of defects. The methods can
be used to visualize defects in many types of materials and can be
applied, for example, for the development and manufacturing of thin
film gas diffusion barriers for the organic light-emitting diodes
(OLEDs), photovoltaic (PV), and liquid crystal display (LCD)
industries as well as the packaging of: medical devices, sensor
skins, electronic circuits, micro-, and nano-systems.
[0051] Kits
[0052] An additional embodiment of the invention is directed
towards kits useful for the detection of surface defects in a
material. The kits can comprise at least one lipophilic fluorescent
substance (e.g., dyes, fluorescent microspheres, or quantum dots)
as described above. The kits can comprise instructions for
performing the above described methods. The kits can comprise a
"positive control" material containing at least one surface defect.
The kits can comprise a "negative control" material that does not
contain surface defects. The kits can comprise a wash material
useful for removing non-localized fluorescent substance from the
material.
[0053] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor(s) to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the scope of the
invention.
EXAMPLES
Example 1
Binding of Lipophilic Fluorescent Materials to Polymers
[0054] A piece of 2 cm.times.2 cm sized thin polyethylene
terephthalate (PET) plastic film coated on one side with alumina.
About 3 mm-wide edge on the lift side of the film is not coated.
The film was immersed in to a solution of 1,3-dimethyl-5-styryl
BODIPY (Invitrogen Corp., Carlsbad, Calif.; 0.04 mg/mL in 70%
ethanol, 30% deionized water (v/v) for 3 minutes. The film was
removed and washed with 70% ethanol (v/v) three times and examined
with a Nikon fluorescence microscope with a 550 nm excitation/570
nm emission filter set. The uncoated edge of polymer membrane
became red fluorescent; the alumina coating was not stained.
Example 2
Detection of Surface Defects Using Dyes
[0055] An equilateral triangle-shaped scratch (about 1 mm.times.1
mm.times.1 mm; the scratch lines had a width of about 0.1 mm) was
made on a piece of alumina coated thin polyethylene terephthalate
(PET) plastic film to mimic a coating defect. The film was immersed
in to a solution of 1,3-di-n-propyl BODIPY (Invitrogen Corp.,
Carlsbad, Calif.; 0.04 mg/mL in 70% ethanol, 30% deionized water)
for 2 minutes. The film was removed and washed with 70% (v/v)
ethanol three times and examined using a Nikon fluorescence
microscope using a standard FITC filter set (490 nm excitation/515
nm emission). The scratch became green fluorescent based on the
deepness of the damage. The remaining unscratched alumina coating
was not stained. This result indicated that the defect in a large
alumina coated film can be 1) labeled with fluorescent dye, 2)
easily detected and 3) easily located.
Example 3
Detection of Surface Defects Using Fluorescent Microspheres
[0056] Several scratches (about 2 mm long and 0.02 mm wide) were
made by scratching a piece of alumina-coated thin polyethylene
terephthalate (PET) membrane with a sharp 22 gauge syringe needle
to mimic a defect.
[0057] A suspension of 20 nm green fluorescent microspheres
(emission maximum at 515 nm) with low surface charge was prepared
by adding 2% microsphere stock to a 50% ethanol, 50% deionized
water mixture (v/v) for a final concentration of 0.5%
microsphere.
[0058] The membrane sample was immersed into the microsphere
suspension for 3 minutes at room temperature. The film was removed
and washed with 50% ethanol (v/v) three times. The membrane was
allowed to dry in air, and was examined using a Nikon fluorescence
microscope with a FITC filter set (excitation: 490 nm/emission: 515
nm). The scratches became bright green fluorescent, and were easily
visualized and localized on the membrane.
Example 4
Detection of Surface Defects Using Dually Fluorescent
Microspheres
[0059] Several scratches (about 2 mm long and 0.02 mm wide) were
made by scratching a piece of alumina-coated thin polyethylene
terephthalate (PET) membrane with a sharp 22 gauge syringe needle
to mimic a defect.
[0060] A suspension of 110 nm dual emission microspheres (emission
max at 565 nm and 755 nm) with low surface charge was prepared by
adding 5% microsphere stock to a 50% ethanol, 50% deionized water
mixture (v/v) for a final concentration of 0.5% microsphere.
[0061] The membrane sample was immersed into the microsphere
suspension for 3 minutes at room temperature. The film was removed
and washed with 50% ethanol (v/v) three times. The membrane was
allowed to dry in air, and was examined using a Nikon fluorescence
microscope with a XF101 filter set (excitation: 543 nm/emission:
565 nm) and a XF48-2 filter set (excitation: 635.+-.25 nm/emission:
725 nm long pass). The scratches became orange fluorescent when
examined under the XF101 filter, and became red fluorescent when
examined under the XF48-2 filter set.
Example 5
Visualization of Mechanical Cracks
[0062] Mechanical cracks were visualized using a diaza-indacene
fluorophore with hydrophobic substituents. 25 nm thick ALD alumina
barrier films were deposited onto polyethylene naphthalate (PEN)
substrates (Teonex Q65, Dupont Teijin, Inc.). The coated specimens,
some of which were mechanically manipulated in order to
intentionally generate defects, were then soaked in a fluorescent
tag solution for 5 min. Solvent solution containing 70% ethanol and
30% water was used to wash away excess tag molecules not attached
to the film. The sample was then dried using clean dry air and
maintained in an ultraviolet-safe environment. A LSM 510 confocal
microscope (Carl Zeiss, Inc.) was used for inspection of the tagged
sample. A 488 nm Argon 12 laser source was used to excite the tags
and the fluorescent emission (maximum at 515 nm) was measured with
a 505-530 nm band pass filter. A PEN substrate with no coating, a
PEN substrate with an ALD alumina coating, and an identically
coated PEN substrate bearing intentionally-made scratches were
compared. The tag molecule attached well to the bare PEN film,
yielding a bright field across the whole sample, whereas an all
dark field image revealed that the tag did not attach to the ALD
alumina (data not shown). FIG. 1 shows that the fluorescent
molecule selectively attached only to the hydrophobic PEN
substrate, where it is exposed by the scratch in the hydrophilic
ALD alumina coating.
Example 6
Visualization of Mechanical "Channel Cracks"
[0063] The failure mode of "channel cracking" is commonly
encountered when a brittle inorganic coating is subjected to
mechanical strain or thermal cycling. However, a series of such
cracks is not readily observed in transparent films. To demonstrate
the use of lipophilic fluorescent tags, an external tensile loading
was applied to PEN substrates coated with 25 nm of ALD alumina. The
fluorescent tags were then applied to these specimens according the
procedure described in Example 5. FIG. 2 shows cracks identified
across the gage section of a specimen that was elongated to 5%
strain. Such cracks, which propagated in the direction orthogonal
to the applied load, are common when the stress in brittle films
exceeds their critical threshold limit. The cracks in FIG. 2A may
be distinguished from those at the edges of the specimen, shown in
FIG. 2B, which were generated during the sample preparation.
Specifically, these edge-located cracks were generated when the
alumina ACD coated specimen was cut to size prior to testing. FIG.
2B identifies the unique characteristics of the shear cracks, which
quickly arrest near the edge of the specimen. Excellent image
contrast was obtained in all of the confocal measurements, allowing
cracks to be readily identified despite minimal sample preparation.
Field emission scanning electron microscopy (FESEM) was used to
measure the width of the cracks. At the fully formed region of the
shear cracks, the crack width of about 20 nm was observed using a
JSM-7401F field emission scanning electron microscope (JEOL
Limited), FIG. 2C.
Example 7
Visualization of Individual Defects and Particles
[0064] In contrast to mechanical cracks, individual defects or
pinholes are generally caused by particulate contamination and/or
the substrate surface roughness. Tiny individual defects in
submicron/nanoscale size are the critical features limiting barrier
performance. These defects have to be inspected and controlled to
assure the barrier quality and high yield barrier manufacturing.
ALD alumina barrier films were deposited onto PEN substrates and
treated with fluorescent tags, as described in Example 5. FIG. 3A
is an image collected using a confocal microscope with a 20.times.
objective showing a defect rich region in the 25 nm thick
Al.sub.2O.sub.3 ALD film. White arrows in FIG. 3A indicate
prescribed marker features, used to facilitate defect location for
further FESEM imaging. To verify the individual defects and
determine the defect size, sites #1 and #2, shown in FIGS. 3B and
3C, respectively, were subsequently observed using FESEM. Diameters
of .about.200 nm and .about.1.2 .mu.m were determined for sites #1
and #2, as indicated in FIGS. 3B and 3C. However, defects smaller
than 200 nm also were rendered visible by treatment with the
fluorescent tag molecule. The images demonstrate that defect sizes
between tens and hundreds of nanometers can be readily visualized
after treatment with the lipophilic fluorescent tag substance. FIG.
3 also provides information about the morphology of the individual
defects. From FIG. 3B, the oval shaped defect bears a tiny crack at
its top end. The defect in FIG. 3C shows a region where the
Al.sub.2O.sub.3 ALD film was not able to bind to the polymer
surface, likely the result of particle contamination. Although the
identified defects possibly could be observed using SEM, defect
inspection becomes very cumbersome because the defect location as
well as the defect density cannot be determined at low
magnification. Further, examination at high magnification with
small field size is very time-consuming. Compared with SEM and AFM
observation, visualization of lipophilic, fluorescent substances
according to the present method allows examination of a large field
size and offers the advantage of continuous inspection at low
magnification.
[0065] All of the compositions and/or methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and/or methods and in
the steps or in the sequence of steps of the methods described
herein without departing from the concept and scope of the
invention. More specifically, it will be apparent that certain
agents which are chemically related may be substituted for the
agents described herein while the same or similar results would be
achieved. All such similar substitutes and modifications apparent
to those skilled in the art are deemed to be within the scope and
concept of the invention.
[0066] All of the U.S. patents, U.S. patent application
publications, U.S. patent applications, foreign patents, foreign
patent applications and non-patent publications referred to in this
specification are incorporated herein by reference, in their
entirety. Aspects of the embodiments can be modified, if necessary
to employ concepts of the various patents, applications and
publications to provide yet further embodiments.
* * * * *