|United States Patent
|Gorfinkel , et al.
||October 15, 2002 |
Multicapillary bundle for electrophoresis and detection for DNA
A multichannel electrophoretic cassette structure is disclosed comprising
distinct regions for loading and detection with different spacing between
channels. A method and an apparatus are further disclosed enabling multicolor
fluorescent detection from a non-coplanar bundle of multiple channels. A method
for fabricating monolithic multichannel cassettes for electrophoresis and
fluorescent detection is also described.
||Gorfinkel; Vera (Stony Brook, NY);
Gouzman; Mikhail (Lake Grove, NY); Serge; Luryi (Old Field,
||State University of New York at Stony
Brook (Stony Brook, NY) |
||December 3, 1999|
|Current U.S. Class:
||204/600; 204/601; 204/603;
204/604; 204/452; 436/63; 356/344; 250/458.1 |
|Field of Search:
356/344 250/458.1 |
References Cited [Referenced
U.S. Patent Documents
||Smith et al.
||Yeung et al.
||Dovichi et al.
||Lauer et al.
||Nasu et al.
||Melman et al.
||Takahashi et al.
||Allbritton et al.
Examiner: Warden; Jill
Assistant Examiner: Sines; Brian
Attorney, Agent or Firm: F. Chau & Associates, LLP
The U.S. Government has a license in
this invention and the right in limited circumstances to require the patent
owner to license others on reasonable terms of grant number HG01487 awarded by
the National Institute of Health (NIH).
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims
priority to Provisional Application Serial No. 60/110,712 filed Dec. 3, 1998 and
incorporated herein by reference.
What is claimed is:
1. A multichannel electropheretic cassette
a plurality of capillaries
arranged in a non-planar bundle;
a loading region for loading biological
molecules into the capillaries;
an observation region for observing
biological molecules in said plurality of. capillaries;
of capillaries extending in an arc between said loading region and said
a photodetector arranged in a first plane inclined
at an angle relative to an axis of the plurality of capillaries at said
said photodetector simultaneously imaging each
capillary in the non-planar bundle, enabling multicolor fluorescent detection of
biological molecules in said plurality of capillaries.
multichannel electrophoretic cassette structure of claim 1, further comprising
known capillary patterns for give cross-sections of the arc.
multichannel electrophoretic cassette structure of claim 1, wherein the loading
region comprises a first cross-sectional dimensionality and the observation
region comprises a second cross-sectional dimensionality.
multichannel electrophoretic cassette structure of claim 3, wherein the
capillaries have a known variable cross-section organization between the loading
region and the observation region.
5. The multichannel electrophoretic
cassette structure of claim 1, wherein the housing includes a heat conducting
6. The multichannel electrophoretic cassette structure of claim
1, wherein the housing includes a means for thermal control of the capillaries.
7. The multichannel electrophoretic cassette structure of claim 1,
further comprising an alignment marker detectable in a cross-section of the
8. The multichannel electrophoretic cassette structure
of claim 7, wherein the alignment marker is a fluid in a predetermined capillary
of the capillary bundle.
9. The multichannel electrophoretic cassette
structure of claim 1, further comprising an illuminator proximate to the
observation region at an angle to the photodetector.
The present invention relates to a method and
apparatus for DNA electrophoresis and detection.
Electrophoretic lanes are widely used for separating multi-component
samples ranging from small inorganic ions to large biological molecules. DNA
electrophoresis is commonly performed with polyacrylamide gel placed between two
glass plates. In recent years, the method of capillary electrophoresis has been
developed, which alleviates the dissipation of Joule heat and permits the
application of higher voltage, thus speeding up the electrophoresis separation
process. In capillary electrophoresis, a buffer-filled capillary is suspended
between two reservoirs filled with a buffer liquid. An electric field is applied
between the two ends of the capillary. The potential difference that generates
the electric field is in the range of kilovolts. Multi-component samples are
typically injected under the influence of an electrical field. The samples
migrate under the influence of electric field, with components of the sample
being electrophoretically separated. After the separation, the components are
detected by a detector.
One of the important applications of
electrophoretic separation is for DNA sequencing. The use of capillary
electrophoresis has improved DNA sequencing rates. Part of the improvement in
speed, however, was initially offset by the loss of the ability (inherent in
slab gels) to accommodate multiple lanes in a single run. Highly multiplexed
capillary electrophoresis, by making possible hundreds or even thousands of
parallel sequencing runs, offers an attractive approach to overcoming the
current throughput limitations of DNA sequencing instrumentation. Typically, an
array of capillaries is held in a guide and the intake (cathode) ends of the
capillaries are dipped into vials that contain samples. After the samples are
taken in by the capillaries, the ends of the capillaries are removed from the
sample vials and submerged in a buffer which can be in a common container or in
The currently used multichannel electrophoretic arrays
typically represent a coplanar arrangement of capillaries. This geometry has
been chosen because of its convenience for detection, which is typically
performed with the help of fluorescent tags (fluorophores) attached to the DNA
fragments migrating along the electrophoretic lanes. The detection is typically
effected by illuminating the lanes within a specially provided translucent
portion near their anode end (the observation region) with a laser source that
excites fluorescence. One of the common reasons for the conventional planar
arrangement of the capillaries has been that it offers a straightforward way of
positioning the photoreceiving matrix that detects the fluorescence from all
lanes in parallel. Another common reason for the parallel arrangement of
capillaries is due to the need for color resolution of different fluorescent
markers, which is typically performed by spatially dispersing the emitted
fluorescent radiation in the longitudinal (along the lanes) direction. The
spatially dispersed radiation from all observation regions is then imaged onto a
two-dimensional photoreceiving matrix, such as CCD or CMOS, using a
high-aperture projection objective. Still another common reason for the parallel
arrangement of capillaries is associated with the desire to illuminate all lanes
at once with a laser beam, which propagates in the plane of the capillaries and
at the same time transverse to their axes.
In recent years, several
authors disclosed such multicapillary systems, see e.g., Quesada et al.,
"Multiple capillary DNA sequencer that uses fiber-optic illumination and
detection", Electrophoresis, vol. 17, pp. 1841-1851 (1996). Moreover,
multicapillary systems have been disclosed in which the capillaries themselves
serve as light-guiding elements for the illumination beam, see, e.g., Yeung et
al., "Multiplexed capillary electrophoresis system", U.S. Pat. No. 5,582,70
(1996) and Quesada et al., "Multi-capillary optical waveguides for DNA
sequencing", Electrophoresis, vol. 19, pp. 1415-1427 (1998).
a need exists for a non-planar arrangement of multiple capillary electrophoretic
lanes which provide miniaturization of the electrophoretic carrier and which
will significantly reduce the cost of multiple-lane DNA sequencing machines. A
further need exists for a method for manufacturing monolithic cassettes,
including multiple capillary lanes and a method and apparatus for parallel
detection of fluorescent markers passing through the observation regions in a
non-planar arrangement of multiple electrophoretic lanes.
The present disclosure describes a non-planar arrangement of multiple
capillary electrophoretic lanes, a technique for manufacturing monolithic
cassettes, comprising such multiple capillary lanes and a method and apparatus
for parallel detection of fluorescent markers passing through the observation
regions in a non-planar arrangement of multiple electrophoretic lanes. The need
for non-planar arrangement arises from the desire to miniaturize the
electrophoretic carrier, which will significantly reduce the cost of
multiple-lane DNA sequencing machines.
The present disclosure offers
inventive solutions that circumvent all of the above-cited common reasons for
choosing co-planar geometry of a multilane assembly. In the simplest embodiment,
the photoreceiving matrix is arranged in a first plane inclined at an angle
relative to the capillary axes, while the observation regions of different
capillaries are arranged in a second plane which is also inclined at an angle
relative to the capillary axes. For example, the first and second planes are
parallel to each other inclined at 45 degrees relative to the capillary axes.
The simultaneous illumination of multiple capillary lanes is effected by an
array of modulated laser sources whose beams have a specially chosen spatial
arrangement and direction relative to the capillary axes and to said first and
second planes. Next, the need for spatial dispersion of fluorescent radiation
into components corresponding to different fluorescent wavelengths is eliminated
in accordance with the method for multicolor fluorescent detection recently
disclosed by Gorfinkel et al., "Method and apparatus for identifying
fluorophores", U.S. Pat. No. 5,784,157 (1998). Further, the need for waveguiding
the incident radiation in the inventive method is substantially eliminated by
using tightly packed capillaries of small cross-section. In a preferred
embodiment, the capillaries have a rectangular or square cross-section of less
than about 100 .mu.m on the side. For example, a rectangular array of 96 such
capillaries has an overall cross-section of less than 1 mm.sup.2. As many as one
thousand capillary lanes can be accommodated in a monolithic array of square
cross-section about 3.times.3 mm. The present invention further discloses
techniques for fabricating such multicapillary arrays. These techniques employ
drawing a glass preform that has a pre-fabricated set of holes of desired shape
(e.g., rectangular) and is similar to drawing hollow optical fibers or glass
ferrules, see, e.g., MacChesney et al., "Materials development of optical
fiber", Journal of the American Ceramic Society, vol. 73, pp. 3537-3556 (1990)
and Anderson et al., "Optical fiber connector comprising a glass ferrule, and
method of making same", U.S. Pat. No. 5,598,496 (1997). In one preferred
embodiment, the preform is prepared with multiple holes to draw a monolithic
multicapillary structure. In another preferred embodiment, a multicapillary
bundle is fabricated by gluing or soldering together a multiplicity of single
Still another aspect of the present invention pertains to
loading tightly packed monolithic capillaries. In one of the preferred
embodiments, this is provided by matching the capillary array cross-section with
a similar array of charging pins on a silicon chip. In another preferred
embodiment, the capillary assembly, which is monolithic in the observation
region near the anode, is made loose like a brush near the cathode end. A
special fixture holder is further provided that fixes the loose cathode ends of
capillaries in a desired pattern. In a preferred embodiment, the loose cathode
ends of the capillaries are arranged in a pattern that matches the common 96
well plate widely used in the preparation of biological samples.
DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates the general
structure of a multicapillary cassette;
FIG. 2 illustrates the
cross-section of the capillary assembly near the observation region;
FIG. 3 illustrates the cross-section of the capillary assembly near the
FIGS. 4a-c illustrate the spatial configuration of the
capillary assembly, the illuminator and the photoreceiver in the observation
region. FIG. 4a: side view; FIG. 4b: top view; FIG. 4c: fluorescent image
projected onto a photoreceiving matrix;
FIG. 5 illustrates a preferred
structure of the illumination source;
FIG. 6 illustrates an array of
independently modulated optical sources and coupling of their radiation outputs
into a single optical fiber;
FIG. 7 illustrates the structure of a
fiber-optic illumination system to provide independently modulated and
reconfigurable optical beams;
FIG. 8 illustrates the structure of a
fiber-optic illumination system with a multiple independent light sources;
FIG. 9 illustrates illumination of capillaries with the help of an
optical line generator;
FIGS. 10a-b illustrate exemplary spatial
arrangements of the capillaries, the optical source and the photoreceivers;
FIG. 11 illustrates the reception of fluorescent signal by a
two-dimensional photoreceiving matrix;
FIG. 12 illustrates the reception
of fluorescent signal by a linear photoreceiving array; and
illustrates the reception of fluorescent signal by a wide area photoreceiver.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
bundle may be implemented either as a monolithic or quasi-monolithic structure
or a loose assembly of individual capillaries. Monolithic structures may be
obtained by drawing on a preform. Quasi-monolithic structures may also be
obtained by a tight packing together of individual capillaries or smaller
monolithic multicapillary units. We also envision intermediate structures, which
may be monolithic or quasi-monolithic in one region and loose in another.
Various versions of the multicapillary bundle can be characterized by
the geometry of their cross-section at different positions along the capillary
lanes, cf. FIG. 1. The geometry of the bundle is important in that it affect the
way the operations of fluorescent detection and sample loading are performed. In
every preferred embodiment, the cross-section of the multicapillary bundle in
the region of detection is monolithic or quasi-monolithic and is characterized
by a definite known pattern of capillaries of a desirable shape. For example
said pattern may be periodic in two dimensions as illustrated in FIG. 2. The
desirable shape of the cross-section of the individual capillary lanes in the
detection region is determined primarily by the convenience of external
illumination and collection of the fluorescent response. For example, said shape
is rectangular or oval but it may also be hexagonal or some other polygonal
shape. The shape of the bundle cross-section and that of individual capillaries
need not be the same in other regions of the bundle as determined by the
convenience of loading, efficiency of electrophoresis and facility of
In one preferred embodiment, the entire bundle is
monolithic or quasi-monolithic. The loading end surface of the bundle may
represent a flat surface perpendicular or inclined to the capillary axis. The
surface may also be processed, e.g., mechanically or chemically, resulting in
non-flat surface. In this embodiment the bundle cross-section may be constant or
variable along the length of the bundle.
In another preferred
embodiment, the bundle is monolithic or quasi-monolithic in the detection region
while loose in the loading region, as illustrated in FIG. 3. Loose capillary
ends are fixed in a pattern determined by a specially provided fixture plate. In
a preferred embodiment, this pattern matches the common 96-well plate containing
DNA samples. Holes in the fixture plate are of desired shape, e.g., cylindrical,
conical, or pyramidal, designed to tightly hold the capillaries. Fixation of the
capillaries in the fixture plate can be done in a variety of ways, e.g., by
gluing or soldering.
Loading of the DNA samples
into the multicapillary bundle can be done by using a variety of known
techniques employed for the injection of DNA samples into single capillary
lanes. These techniques include, e.g., the mechanical transfer and
electro-kinetic injection. The inventive techniques disclosed here relate to
specific configurations that facilitate loading into a bundle of capillaries.
Firstly, the loading device must be adapted to the cross-sectional dimensions of
the bundle in the loading region. The preferred geometry of the loading device
comprises one or more adapters that have a similar pattern as the bundle
cross-section. Said adapters may be attached to the source of DNA samples, such
as a multi-well plate or a micro-fluidic chip. Said adapters may also be
attached to the capillary bundle in a removable or permanent fashion. The
adapter may comprise a pattern of holes or protuberances that fit the capillary
pattern. Connection between the adapter and the capillary pattern may be either
male-to-female or female-to-male. Alternatively, the adapter may be elastic and
have a flat surface with holes so that a tight connection is established simply
by pressing the edge of the capillary bundle on the adapter.
device may provide means for electrokinetic injection. To this end, it must be
outfitted with one or more electrodes. The controlling voltage may be applied to
different electrodes individually, so that different voltages are applied to
Referring to FIG. 1, spatial arrangement of
elements of a preferred embodiment of the multicapillary cassette for DNA
housing 11; multicapillary bundle 12; observation
region 13; and loading region 14.
In FIG. 1, it is assumed that the
anode and the cathode are placed outside the housing 11 so that capillaries
continue beyond regions 13 and 14. The housing volume may be filled with a heat
conducting fluid or other means for thermal control of the capillaries.
Referring to FIG. 2, a cross-section of the capillary assembly near the
observation region is shown. The M.times.N array comprises rectangular
capillaries arranged in M columns and N rows. The shape of capillaries can be
rectangular, square, elliptic, or any other selected for the convenience of
illumination and collection of fluorescence. The capillary assembly in this
region is a tightly packed bundle. In a preferred embodiment the assembly is
monolithic obtained by drawing a preform with multiple holes of desired shape.
In another preferred embodiment the assembly is made up of single, for example,
rectangular capillaries, soldered or glued together using solder or glue of
properly matched refractive index.
Referring to FIG. 3, a cross-section
of the capillary assembly near the loading region is shown. Loose capillary ends
are fixed in a pattern determined by the fixture plate. In a preferred
embodiment, this pattern matches the common 96-well plate containing DNA
samples. Holes in the fixture plate are of desired shape, e.g., cylindrical,
conical, or pyramidal, designed to tightly hold the capillaries. Fixation of the
capillaries in the fixture plate can be done in a variety of ways, e.g., by
gluing or soldering. A thermal process based on the thermal expansion and
contraction of the holes can also be used. In another preferred embodiment the
capillary ends are not loose but are monolithic, for example, obtained by
drawing on a preform. In such embodiments it is contemplated that the well plate
from which samples are injected into capillaries is implemented as a microchip
or a micro-assembly to match the miniature cross-section pattern of a monolithic
The arrangement of capillaries in a
cross-section of the capillary assembly near the loading region may be organized
in a different way from that near the observation region. While the total number
of capillaries is obviously the same in both cross-sections their row x column
pattern may be quite different. For example, one may still have a matrix of
dimensions P.times.Q=M.times.N, where M and N refer to FIG. 2, but the factors
P,Q are different from M,N.
To facilitate precise manipulation of the
capillary bundle and its alignment relative to the loading device, special set
of alignment marks may be provided, that is clearly visible or detectable in a
cross-section of the bundle in the loading region. These marks may employ an
optical or some other physical effect. In one preferred embodiment, the desired
set of alignment marks is obtained by filling a reserved group of capillaries in
the bundle with some easily detectable material. For example, said group of
capillaries may be filled with some conducting or magnetic fluid, or some
distinguishable optically contrast fluid, such as containing color luminescent
or fluorescent species.
Referring to FIGS. 4a-c, an illustration of the
spatial configuration of the capillary assembly, the illuminator and the
photoreceiver in the observation region, is shown. FIG. 4a shows a side view of
the relevant portion of the apparatus. FIG. 4b shows a top view of the relevant
portion of the apparatus. FIG. 4c shows a planar view of the fluorescent image
projected onto the target screen of the photoreceiving system. FIGS. 4a-c
include: assembly (bundle) of capillaries 41; focal plane of the optical
receiving system 42; one of the capillaries of the assembly 43; fluorescent zone
44 in one of the capillaries 43 of the assembly; optical receiving system 45,
such as projection optics; photoreceiving system 46, such as CCD or CMOS, or PMT
matrix; image of the fluorescent zones 47 on the target screen of the
photoreceiving system 46; optical axis 48 of the optical receiving system with
the angle between said optical axis and the capillary axes denoted by .alpha.,
for example, .alpha.=45.degree.; one of the optical paths 49, including
projection optics, carrying the excitation beam from illumination sources.
The illumination sources are arranged so that the optical excitation
beams they emit propagate in the focal plane 42 of the optical receiving system
45. Said excitation beams need not be parallel to each other but may be
parallel. In a preferred embodiment, illustrated in FIG. 4a, the optical
excitation beams propagate perpendicular to a plane containing a row of
capillaries, i.e., perpendicular to the cross-section of the assembly displayed
in the plane of FIG. 4a. In FIG. 4b the direction of illumination beams lies in
the plane of the drawing and in the direction of sources 49. The image 47 on the
target of the photoreceiver 46 is shown in FIG. 4c as a plane view.
facilitate the spatial alignment of the capillary assembly, the illuminator and
the photoreceiver in the observation region, the capillary bundle may be
outfitted with alignment marks clearly visible or detectable in a cross-section
of the bundle in the observation region, such as plane 42. For example, said set
of markers may be obtained by reserving several capillaries in the bundle to be
filled with some distinguishable fluorescent fluid or fluids.
to FIG. 5, an illustration of the spatial arrangement of elements of the
illumination system are shown and may include: an optical channel 51 delivering
the desired combination of modulated spectral components from the optical source
and a narrow excitation beam 52 directed onto the capillary assembly.
FIG. 5 displays separately a portion of FIG. 4c to illustrate the
possibility of implementing the illumination system as a group of independent,
not necessarily parallel, optical systems, each comprising a modulated source.
In another preferred embodiment, illustrated in FIG. 7, the illumination is
obtained from a single multiplexed optical source. In general, the number of
independent optical sources can be smaller than, equal to or large than the
number of illumination channels 52.
Referring to FIG. 6, an illustration
of an array of independently modulated optical sources coupled into a single
optical path, for example, an optical fiber and may include: an optical coupler
61; optical channels 62, e.g., fibers, delivering modulated narrow-band optical
spectral component to coupler 61; modulated narrow-band light source 63, e.g.,
diode laser, LED, or a gas or solid-state laser with an external modulator;
electrical signals 64 modulating light sources 63, for example, at distinct
radio frequencies f.sub.i ; the source of modulating signals 65, e.g., a
modulated current driver for laser diode.
The narrow-band optical
spectral components (.lambda..sub.1, . . . , .lambda..sub.4) independently
modulated at distinguishable radio frequencies (f.sub.1, . . . , f.sub.4) are
delivered to the inputs of the optical coupler 61 which combines these
components into a single optical path 51, for example an optical fiber.
Referring to FIG. 7, an illustration of a fiber-optical illumination
system delivering a multiple independently modulated and reconfigurable optical
beams may include: an optical demultiplexer or beam splitter 71; modulating
electrical signals 72 at distinct radio frequencies; optical modulators 73,
e.g., choppers, controlled by signal 72. Optical beam 51 containing multiple
spectral components coupled into a single optical path is delivered to the input
of a beam splitter 71 which provides at its multiple outputs a number of optical
beams. These beams may or may not be similar in intensity or polarization. Each
beam is modulated independently by modulators 73 controlled by electrical
Referring to FIG. 8, an illustration of a fiber-optic
illumination system with a multiple independent light sources is shown. Each of
the narrow optical excitation beams 52 directed onto the capillary assembly
comprise multiple modulated optical spectral components taken from the optical
channel 51 which delivers the desired combination of modulated spectral
components from the optical coupler 61. As illustrate in FIG. 6, said optical
coupler 61 gathers multiple spectral components from a set of modulated light
sources 63 and couples them into a single optical channel 1, for example an
Referring to FIG. 9, an illustration of capillary bundle
illumination with the help of an optical line generator is shown which includes:
an optical line generator 91; a divergent asymmetric beam of light 92; an
asymmetric beam collimator 93; and a collimated laterally extended optical
illumination beam 94. Optical line generator is inserted in the beam path before
the capillary assembly. The narrow optical excitation beam 52 is transformed by
the optical line generator 91 into a divergent asymmetric beam 92. The asymmetry
of the beam means that the beam cross-section is highly asymmetric, e.g.
elliptic rather than circular. In the plane where the divergent beam reaches
collimator 93, said beam is extended in one direction so as to illuminate the
full section of the multi-capillary assembly. In the other direction the beam
remains as narrow as possible, preferably close to the original width of beam
52. The purpose of the collimator 93 is to transform the divergent beam 92 into
a parallel (collimated) beam 94.
Another preferred embodiment of an
optical line generator is to provides means for scanning the beam 52 laterally
over the full section of the multi-capillary assembly. In contrast to the
conventional beam scanners which scan by changing the angular direction of a
pencil beam, the scanned beam according to present invention is obtained by
parallel transfer of a pencil beam, retaining the same angular orientation. Such
scanning means are well known to those skilled in the art.
FIGS. 10a-b, an illustration of exemplary spatial arrangements of the capillary
bundle relative to the optical source and the photoreceiver are shown and
include: a direction along an electrophoretic lane 101 indicating the average
motion of labeled DNA fragments. FIG. 10a shows an arrangement similar to FIG.
4a except that the narrow optical excitation beam 52 is incident on the
capillary assembly 41 at an oblique angle. The beam 52 and optical paths 49
represent a whole plane of beams 52 and paths 49 which in the drawing 10a is
perpendicular to the plane of the drawing. Similar representation is assumed in
FIG. 10b which shows the same elements as FIG. 10a but arranged at still another
relative orientation. In FIG. 10b the plane of beams 52 and paths 49 is
perpendicular to capillary axes (direction 101). The projection optics 4 is
oriented so that the photoreceiving system 46 receives the image of a plane
perpendicular to direction 101.
Referring to FIG. 11, reception of the
fluorescent signal by a two-dimensional photoreceiving matrix is shown and
includes: one pixel of a two-dimensional photoreceiving matrix 111. The electric
output of each pixel represents a set of amplitudes A.sub.j (f.sub.j) of
received optical signal at radio frequencies of modulation.
FIG. 12, reception of the fluorescent signal by a linear photoreceiving array
includes one pixel of the linear photoreceiving array 121 and a projection of a
single fluorescent spot 122 from a single capillary element of one the N
capillary columns (see FIG. 2).
Referring to FIG. 13, reception of
fluorescent signals by a wide area photoreceiver includes a target 131 of wide
area photoreceiver and a projection 132 of a single fluorescent spot from a
single capillary element of the M.times.N capillary bundle (FIG. 2).
Commonly assigned provisional applications U.S. application Ser. No.
60/110,714 and 60/110,720 are incorporated herein by reference.
described preferred embodiments of a system and method of the invention (which
are intended to be illustrative and not limiting), it is noted that
modifications and variations can be made by persons skilled in the art in light
of the above teachings. It is therefore to be understood that changes may be
made in the particular embodiments of the invention disclosed which are within
the scope and spirit of the invention as outlined by the appended claims. Having
thus described the invention with the details and particularity required by the
patent laws, what is claimed and desired protected by Letters Patent is set
forth in the appended claims.
* * * * *