U.S. patent application number 10/055517 was filed with the patent office on 2003-07-24 for methods for fluorescence detection that minimizes undesirable background fluorescence.
Invention is credited to Reel, Richard T..
Application Number | 20030136921 10/055517 |
Document ID | / |
Family ID | 21998374 |
Filed Date | 2003-07-24 |
United States Patent
Application |
20030136921 |
Kind Code |
A1 |
Reel, Richard T. |
July 24, 2003 |
Methods for fluorescence detection that minimizes undesirable
background fluorescence
Abstract
The present invention provides a method for the excitation of a
fluorescent sample and the measurement of the fluorescent emission.
The method of the present invention has the advantage of
significantly reducing the amount of background fluorescence. The
method includes the steps of exciting a sample in a substrate with
a beam of light that enters the substrate at an angle less than or
equal to 45.degree. C., and preferably, less than or equal to
20.degree., and then collecting the fluorescent emission form the
sample with a lens system which focuses the emitted light onto a
CCD for detection. Although the following description of the
present invention uses a scanning system using channel plates by
way of illustration, the method described herein may also be used
with non-scanning systems as well as capillary systems.
Inventors: |
Reel, Richard T.; (Hayward,
CA) |
Correspondence
Address: |
HARNESS, DICKEY & PIERCE, P.L.C.
P.O. BOX 828
BLOOMFIELD HILLS
MI
48303
US
|
Family ID: |
21998374 |
Appl. No.: |
10/055517 |
Filed: |
January 23, 2002 |
Current U.S.
Class: |
250/458.1 |
Current CPC
Class: |
G01N 21/645 20130101;
G01N 2021/6495 20130101; G01N 2021/6482 20130101 |
Class at
Publication: |
250/458.1 |
International
Class: |
G01N 021/64 |
Claims
What is claimed is:
1. A method for detecting the fluorescence of a fluorescent sample
in a channel plate having a channel axis comprising: exciting the
fluorescent sample with an excitation beam of light, wherein the
excitation beam of light enters the sample at an angle less than or
equal to about 45.degree. longitudinal axis of the channel axis;
and collecting the fluorescence of the sample with a collection
optics system, wherein the collection optics system collects the
fluorescence and refocuses the fluorescence onto a detector.
2. The method as defined in claim 1, wherein the detector comprises
a detector selected from the group consisting of charge coupled
devices, CMOS detectors, photodiode, photodiode array,
photomultiplier tubes, photomultiplier tube arrays.
3. The method as defined in claim 1, wherein the collection optics
system collimates the fluorescence and refocuses the fluorescence
onto a detector.
4. The method as defined in claim 1 wherein the excitation beam of
light enters the sample at an angle less than or equal to about
20.degree..
5. The method as defined in claim 1 wherein the collection optics
further removes scattered light from the excitation beam using a
long pass filter.
6. The method as defined in claim 1, wherein the collection optics
further removes scattered light from the excitation beam using a
band pass filter.
7. The method as defined in claim 5 further comprising directing
the excitation beam of light substantially parallel to the channel
plate into a reflective mirror, which directs the excitation beam
of light into the sample at an angle less than or equal to about
45.degree..
8. The method as defined in claim 7 further comprising directing
the excitation beam of light from the reflective mirror through a
prism to direct the reflected excitation beam of light into the
sample.
9. An apparatus for fluorescence detection of a sample comprising:
a light source operable to generate an excitation beam of light; a
mirror operable to reflect said excitation beam of light into the
sample at an angle less than or equal to about 45.degree.; and a
collection optics operable to collect fluorescence from the sample
and refocus the fluorescence.
10. The apparatus as defined in claim 9 further comprising a charge
coupled device which receives the refocused fluorescence from said
collection optics.
11. The apparatus as defined in claim 9 wherein said collection
optics further includes a long pass filter operable to remove
scattered light at a wavelength of said excitation beam of
light.
12. The apparatus as defined in claim 9 wherein said collection
optics further includes a transmission defraction grading operable
to separate light into differing wavelengths.
13. The apparatus as defined in claim 9 further comprising a prism
operable to focus said excitation beam of light into the
sample.
14. The apparatus as defined in claim 9 wherein said light source
is a laser.
15. The apparatus as defined in claim 9 further comprising a
channel plate defining a channel which houses the sample.
16. A method for fluorescence detection of a sample comprising:
providing a channel through which the sample is able to flow, the
channel having an axis; directing an excitation beam of light into
the sample at an angle with respect to the axis of the channel;
generating a fluorescence image of the sample and a fluorescence
image associated with the background, the fluorescence image of the
sample being generated in a direction displaced from that of the
fluorescence image associated with the background; collecting
fluorescence of the sample with a collection optics system, wherein
the collection optics system collimates the fluorescence and
refocuses the fluorescence onto a charged coupled device.
17. The method as defined in claim 16 wherein said excitation beam
of light is directed at the sample at an angle less than or equal
to about 45.degree. with respect to the axis of the channel.
18. The method as defined in claim 17 wherein the excitation beam
of light is directed at the sample at an angle less than or equal
to about 20.degree. with respect to the axis of the channel.
19. The method as defined in claim 16 further comprising providing
a laser to generate the excitation beam of light.
20. The method as defined in claim 19 further comprising directing
the excitation beam of light from the laser to a reflective mirror
which directs the excitation beam of light into the sample at an
angle.
21. The method as defined in claim 20 further comprising directing
the excitation beam of light from the reflective mirror through a
prism to direct the reflective excitation beam of light into the
sample.
22. A method of fluorescence detection using a charge coupled
device having a plurality of detector elements, said method
comprising: (a) providing a channel through which a sample can
flow, the channel having a channel axis; (b) directing an
excitation beam of light at an angle with respect to the channel
axis so that the fluorescence image of the sample is generated in a
direction displaced from that of the fluorescence image associated
with background; (c) determining the signal-to-noise ratio of a
first group of detector elements; (d) determining the
signal-to-noise ratio of a second group of detector elements, said
second group of detector elements including at least one detector
element in said first group of detector elements; (e) comparing the
signal-to-noise ratio of the first group of detector elements to
the signal-to-noise ratio of said second group of detector
elements; and repeating steps (c)-(e) with groups of detector
elements of increasing number of detector elements until the
signal-to-noise ratio of said second group of detector elements
declines with respect to the signal-to-noise ratio of said first
group of detector elements.
23. The method of fluorescence detection set forth in claim 22,
further comprising the additional steps of: exciting the
fluorescent sample with an excitation beam of light, wherein the
excitation beam of light enters the sample at an angle less than or
equal to about 45.degree. with respect to the channel axis; and
collecting the fluorescence of the sample with a collection optics
system, wherein the collection optics system collimates the
fluorescence and refocuses the fluorescence onto a charge coupled
device.
24. The method of fluorescence detection set forth in claim 23,
wherein the excitation beam of light enters the sample at an angle
less than or equal to about 20.degree. with respect to the channel
axis.
25. The method of fluorescence detection set forth in claim 22,
further comprising: providing a light source operable to generate
an excitation beam of light; providing a mirror operable to reflect
said excitation beam of light into the sample at an angle less than
or equal to about 45.degree. with respect to the channel axis; and
providing collection optics operable to collimate fluorescence from
a sample and refocus the fluorescence onto the charge coupled
device.
26. The method of fluorescence detection set forth in claim 22,
further comprising collecting fluorescence of the sample with a
collection optics system, wherein the collection optics system
collimates the fluorescence and refocuses the fluorescence onto a
charged coupled device.
27. An apparatus configured to measure fluorescence of a sample
comprising: a source operable to illuminate the sample with an
excitation beam, said excitation beam having a propagation axis
with an incident angle of less then about 45 degrees with respect
to a surface of the sample; collection optics operable to collect
fluorescence from the sample, said collection optics having an axis
which is greater than about 45 degrees with respect to the
propagation axis of said excitation beam.
28. The apparatus according to claim 27, wherein said axis of said
collection optics is oriented about 90 degrees with respect to the
propagation axis of said excitation beam.
Description
FIELD OF THE INVENTION
[0001] This invention relates generally to fluorescence analytical
techniques. More specifically, the invention relates to a method
and apparatus for detecting a fluorescent sample that minimizes
undesirable background.
BACKGROUND OF THE INVENTION
[0002] Fluorescence detection is widely used in biochemical and
medical research applications due to its high sensitivity. For
example, fluorescence detection is used in automated DNA
sequencing, capillary electrophoresis and a variety of
immunoassays. In response to excitation, fluorescent biomolecules
and dyes emit light at characteristic wavelengths, which differ
from the excitation wavelength. By detecting these characteristic
wavelengths, the composition of a sample can be determined.
[0003] In many biological applications, the amount of sample to be
detected is usually quite small. Over the years, methods and
apparatus have been able to manipulate and separate on smaller and
smaller scales, going from the .mu.M range to nM and pM ranges. As
the sample size decreases, the background fluorescence becomes more
significant in relation to the fluorescence of the sample.
[0004] The dominant background noise source in fluorescence
detectors is often shot noise. Shot noise comes from the sample and
background fluorescence. The background fluorescence comes from
fluorescence or Raman scattering from the sample as well as from
the substrate that the sample is contained in. High background
fluorescence also reduces the dynamic range of the detector by
causing saturation of the detector. Therefore, reducing the
background noise is one strategy for improving the performance of
fluorescence detectors.
SUMMARY OF THE INVENTION
[0005] The present invention provides a method for the excitation
of a fluorescent sample and the measurement of the fluorescent
emission that significantly reduces the amount of background
fluorescence. The method includes the steps of exciting a sample in
a substrate with a beam of light that enters the substrate at an
angle less than or equal to about 45.degree., and more preferably,
less than or equal to about 20.degree. and collecting the
fluorescent emission from the sample with a lens system which
focuses the emitted light onto a charge coupled device (CCD) for
detection.
[0006] The beam of light is generated from a laser and is directed
to the sample-containing substrate by a scanning mirror and a
prism. The light enters the substrate at an angle less than or
equal to about 45.degree., and more preferably, less than or equal
to about 20.degree. with respect to the axis of the channel plates
and continues through the channel plate into the sample. In another
embodiment, a lens system collects and collimates the fluorescence
emitted by the excited sample. The collected light then passes
through a wide bandpass filter to exclude scattered laser light.
The collected light then passes through a transmission grating
which disperses the light in the spectral axis, which is oriented
perpendicular to the axis of the substrate. The image is then
focused onto a scientific grade CCD for detection.
[0007] The method of the present invention can be used with a
scanning system or a non-scanning system. Non-limiting examples of
application of the present invention are scanning systems using
channel plates and capillary systems such as capillary
electrophoresis.
[0008] Further areas of applicability of the present invention will
become apparent from the detailed description provided hereinafter.
It should be understood that the detailed description and specific
examples, while indicating the preferred embodiment of the
invention, are intended for purposes of illustration only and are
not intended to limit the scope of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] The various advantages of the present invention will become
apparent to one skilled in the art by reading the following
specification and subjoined claims and by referencing the following
drawings in which:
[0010] FIG. 1 is a schematic block diagram illustrating a
fluorescence detection system; and
[0011] FIG. 2 is a photograph showing the CCD image generated
following exposure to an excitation beam of light.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0012] The present invention provides a method for the excitation
of a fluorescent sample and the measurement of the fluorescent
emission. The method includes the steps of exciting a sample in a
substrate with a beam of light that enters the substrate at an
angle less than or equal to about 45.degree. C., and more
preferably, less than or equal to about 20.degree., and then
collecting the fluorescent emission from the sample with a lens
system which focuses the emitted light onto a CCD for detection.
Although the following description of the present invention uses a
scanning system using channel plates by way of illustration, the
method described herein may also be used with non-scanning systems
as well as capillary systems.
[0013] In one embodiment of the invention, the excitation beam is
directed to the channel by a scanning mirror and prism. The
excitation beam can be generated by a UV, visible or infrared light
source, preferably by a laser. The angle of the mirror can be
adjusted to control the angle that the excitation beam enters the
channel. Preferably, the excitation beam enters the channel at an
angle less than or equal to about 45.degree., and more preferably,
less than or equal to about 20.degree.. The optimal angle for the
incident beam will depend on the index of refraction of the
material of the channel plate. Generally, the more shallow the
angle, the greater the amount of sample fluorescence collected
along with a concomitant reduction in the background
fluorescence.
[0014] In another embodiment of the present invention, collection
optics collects and collimates the fluorescence from the excited
sample into parallel rays. Preferably the lens is situated
perpendicular to the channel axis. The collecting lens may be a
simple camera lens. The collected light then passes through a long
pass or wide bandpass filter, which removes scattered light at the
laser wavelength. The remaining filtered light, which consists
essentially of fluorescence from the sample and background from the
channel, then passes through a transmission detraction grating. The
transmission grating separates the light into rays of differing
wavelength that diverges along the direction of the spectral axis,
perpendicular to the channel axis. Finally a focusing lens directs
the light onto the CCD.
[0015] In a further embodiment, the image from the CCD is collected
to bin or read out as a data file. Preferably, only the section of
the image on the CCD associated with the fluorescence of the sample
is collected by selection of the appropriate pixels to bin and read
out. In the method of the present invention, as the excitation beam
moves through the plate at an angle, the excitation beam creates a
fluorescent trail. When this trail is imaged by the collection
optics, the fluorescence from different parts of the channel will
fall on different sections of the spatial axis of the CCD. As
illustrated in FIG. 2, the fluorescence associated with the sample
is separated from the background fluorescence
[0016] An apparatus for performing the above-described methods of
the present invention is shown in FIG. 1. FIG. 1 illustrates an
apparatus 10 for use with a scanning system using a channel plate
12. The channel plate 12 defines a channel 14 which receives a
medium that contains samples 16. A current is applied to the medium
that contains the samples 16 by means of a pair of electrodes 18.
Upon passing a current through the medium, the samples 16 are
separated as is known in the electrofluorescence art. The channel
plate 12 may be formed from glass, fused silica, plastic or other
transparent type material. The channel plate 12 is supported by a
support plate 13 formed from glass, fused silica, plastic or other
transparent material. In addition, the channel 14 may be defined by
other suitable structures such as capillary tubes, arrays of
capillary tubes and slab gel with field defined lanes.
[0017] A laser 20 generates an excitation beam 22 that is
essentially parallel to the channel plate 12 and directed toward a
reflective mirror 24. The mirror 24 is adjusted to reflect the
excitation beam 22 at the desired angle into the channel plate 12.
Here again, the excitation beam 22 enters the channel plate 12 at
an angle less than or equal to about forty-five degrees
(45.degree.), and preferably less than or equal to about twenty
degrees (20.degree.). The excitation beam 22 is directed through a
prism 26 to facilitate entry of the excitation beam 22 into the
support plate 13. The support plate 13 is optically coupled to the
channel plate 12 using water, direct contact or any transparent
material with an index similar to the channel plate. The focused
excitation beam 22 enters the channel plate 12 and passes through
the channel plate 12 before reaching the channel 14 containing the
sample 16. The focused excitation beam 22 continues through the top
layer of the channel plate 12. As defined by the Fresnel Equations,
some light is reflected at boundaries where the index of refraction
changes. This creates the reflected beams 28. Both the focused
excitation beam 22 and the reflected beam 28 can generate
undesirable fluorescent emissions from the samples 16.
[0018] A portion of the sample fluorescence emissions 30 enters
collection optics 32 where the emitted light is collected,
collimated and dispersed before being focused onto CCD 34 using
known optics and CCD technology. In this regard, the collection
optics 32 includes a first collimating lens, which collects and
collimates the fluorescence from the excited sample 16 into
parallel rays. The collected light is then passed through a long
pass or laser rejection filter, which removes scattered or stray
light at the laser wavelength(s). The remaining filtered light,
which consists of the fluorescence emissions from the sample 16 and
fluorescent background from the channel plate 12 is then passed
through a transmission defraction grating, a grism, a prism or
reflected off a reflective grating. The transmission detraction
grating separates the lights into rays of differing wavelengths
that diverges along the direction of the spectral axis which is
perpendicular to the axis of the channel 14. Finally, a second
focusing lens focuses the light onto the CCD 34. The collection
optics 32 may be similar to the optics system disclosed in, U.S.
Ser. No. 09/564,790 filed May 5, 2000 or Simpson et al., "A
Transmission Imaging Spectrograph and Micro fabricated Channel
System for DNA Analysis", Electrophoresis 2000, 21, 135-149, both
of which are hereby incorporated by reference. However, other
suitable optics systems may be used. Further, it will be
appreciated other types of detectors can also be used to receive
light from the sample 16. These include CMOS detectors,
photodiodes, photodiodes arrays, photomultiplier tubes,
photomultiplier tube arrays or other suitable detectors. In
addition, the preferred orientations of the collection optics 32 is
substantially perpendicular to the excitation beam 22 entering the
sample 16 as this allows a larger amount of light from the sample
to be collected while still rejecting background.
[0019] The image from the CCD 34 is collected to be read out as a
data file as is shown and illustrated in FIG. 2. In this regard,
FIG. 2 illustrates both the glass fluorescence 36, which is the
background fluorescence from the channel plate 12, as well as the
collected light 38 from within the channel 14 which consists of the
fluorescence from the sample 16. This collected light 38 is
preferably from the only section of the image on the CCD 34
associated with the fluorescence of sample 16, which is collected
by selection of the appropriate detector elements or pixels (which
receive little background fluorescence) from which meaningful data
is to be read. The CCD 34 may also use "binning" in which the
photogenerated charge in adjacent detector elements are read out as
a combined charge pocket during a single read. The use of binning
may reduce overall noise when compared to other processing
techniques, but may be accompanied by loss of spacial
resolution.
[0020] By allowing the excitation beam 22 to enter the channel
plate 12 at an angle of less than about forty-five (45.degree.),
and preferably less than or equal to about twenty degrees
(20.degree.), much of the reflected light and background
fluorescence that is produced by the excitation beam 22 entering
the channel plate 12 is directed away from the collection optics as
is shown in FIG. 2. Accordingly, because less background noise
enters the collection optics 32; the sensitivity of the apparatus
10 is increased. In addition, because the excitation beam 22 enters
the plate at a relatively small angle with respect to the
collection plate 12, a greater amount of fluorescent light is
created by the samples 16 causing an increase in the fluorescence
of the sample and therefore improved sensitivity.
[0021] The method for selecting which pixels or detector elements
are used to generate meaningful data from which spectral
information is to be determined, and which pixels or detector
elements should be ignored as receiving excessive background
fluorescence, involves two considerations. The signal-to-noise
ratio of the CCD 34 should be maximized while the dynamic range of
the CCD 34 is not excessively limited. One method for selecting
which detector elements should be used to generate meaningful data
for analysis is as follows.
[0022] First, the output from a first group of detector elements
near the center of the image on the CCD 34 is recorded and the
signal-to-noise ratio determined. Once the signal-to-noise ratio
has been determined from this first set of detector elements, the
signal-to-noise ratio is compared to the signal-to-noise ratio from
a second group of detector elements. This second group of detector
elements includes those detector elements in the first group as
well as detector elements that are adjacent to the detector
elements in the first group. If the signal-to-noise ratio
increases, this indicates that better data can be obtained if the
second group of detector elements is used to generate spectral
information as compared to using the first group of detector
elements. The output of the second group of detector elements is
therefore initially selected to be used to generate meaningful data
from which spectral information is to be determined.
[0023] This process is continued with progressively larger sets of
detector elements until the signal-to-noise ratio begins to
decline. When the signal-to-noise ratio begins to decline, the
inclusion of additional detector elements does not improve
collection of meaningful data and therefore the output from the
remaining detector elements is not considered. However, during this
process, care must be taken that the background noise does not
consume so much of the capacity of the CCD 34 so as to diminish the
dynamic range of the CCD 34. If too much of the dynamic range is
consumed a lower number of bins should be used.
[0024] The foregoing description discloses and describes merely
exemplary embodiments of the present invention. For example, the
excitation beam 22 could enter the channel plate 12 at an angle
greater than 45.degree. if the collection optics 32 is located
off-axis (i.e., off-set from the direction perpendicular to the
direction of the excitation beam 22 entering the sample). Further,
the inside top surface of the channel plate 12 may be coated with a
low index material or fabricated from a low index material (e.g.,
Teflon AF). In such a case, the excitation beam 22 could be
orientated at an angle (i.e., about 22.degree.) that would be
totally internally reflected so as to further separate the
detection region from the background region. Further, multiple
excitation beams 22 may be used as well as multiple detection
elements. One skilled in the art will readily recognize from such
discussion, and from the accompanying drawings that various
changes, modifications and variations can be made therein without
departing from the spirit and scope of the invention.
* * * * *