U.S. patent number 5,091,652 [Application Number 07/531,900] was granted by the patent office on 1992-02-25 for laser excited confocal microscope fluorescence scanner and method.
This patent grant is currently assigned to The Regents of the University of California. Invention is credited to Richard A. Mathies, Konan Peck.
United States Patent |
5,091,652 |
Mathies , et al. |
February 25, 1992 |
**Please see images for:
( Certificate of Correction ) ** |
Laser excited confocal microscope fluorescence scanner and
method
Abstract
A fluorescent scanner for scanning the fluorescence from a
fluorescence labeled separated sample on a sample carrier including
a confocal microscope for illuminating a predetermined volume of
the sample carrier and/or receiving and processing fluorescence
emissions from said volume to provide a display of the separated
sample.
Inventors: |
Mathies; Richard A. (Contra
Costa, CA), Peck; Konan (Contra Costa, CA) |
Assignee: |
The Regents of the University of
California (Oakland, CA)
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Family
ID: |
27040739 |
Appl.
No.: |
07/531,900 |
Filed: |
June 1, 1990 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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463757 |
Jan 12, 1990 |
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Current U.S.
Class: |
250/458.1;
204/612; 250/459.1; 250/461.1; 250/461.2 |
Current CPC
Class: |
G01N
21/6458 (20130101); G01N 27/44721 (20130101); G01N
30/74 (20130101); G02B 21/0076 (20130101); G02B
21/0028 (20130101); G02B 21/004 (20130101); G01N
21/6456 (20130101); G01N 2021/6439 (20130101); G01N
2021/6476 (20130101); G01N 30/95 (20130101) |
Current International
Class: |
G01N
27/447 (20060101); G01N 21/64 (20060101); G02B
21/00 (20060101); G01N 30/74 (20060101); G01N
30/00 (20060101); G01N 30/95 (20060101); G01N
021/64 () |
Field of
Search: |
;250/234,461.2,461.1,459.1,458.1 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Mathies, R. A. and Stryer, L. (1986), Applications of Fluorescence
in the Biomedical Sciences, Eds. Taylor, D. L., Waggoner, A.SA.,
Lanni, F. Murphy, R. F., and Birge, R. (Alan R. Liss, Inc., New
York) pp. 129-140. .
Nguyen, D. C.; Keller R. A.; Jett, J. H.; Martin, J. C. (1987),
Detection of Single Molecules of Phycoerythrin in Hydrodynamically
Focused Flows by Laser-Induced Fluorescence, Anal. Chem. 59,
2158-2161. .
Peck, K.; Stryer, L.; Glazer, A. N.; Mathies, R. A. (1989) Single
molecule fluorescence detection: Autocorrelation criterion and
experimental realization with phycoerythrin Proc. Natl. Acad. Sci.
U.S.A., 86, 4087-4091. .
Mathies, R. A.; Peck, K.; Stryer, L. (1990), Optimization of
High-sensitivity Fluorescence Detection, submitted for publication.
.
Glaser, A. N.; Peck, K.; Mathies, R. A. (1990) A stable
double-stranded DNA--ethidium homodimer complex: Application to
picogram fluorescence detection of DNA in agarose gels, submitted
for publication, Proc. Nat. Acad. Sci. U.S.A. .
Ansorge, W.; Rosenthal, A.; Sproat, B.; Schwager, C.; Stegemann,
J.; & Voss, H. (1988) Non-radioactive automated sequencing of
oligonucleotides by chemical degradation, Nucleic Acids Research
vol. 16, 2203-2207. .
Smith, L. M.; Sanders, J. Z.; Kaiser, R. J.; Hughes, P.; Dodd, C.;
Connell, C. R.; Heiner, C.; Kent, S. B. H. & Hood, L. E.
(1986), Fluorescence detection in automated DNA sequence analysis,
Nature, vol. 321, 674-679. .
Sanger, F.; Nicklen, S.; & Coulson, A. R. (1977), DNA
sequencing with chain-terminating inhibitors, Proc. Natl. Acad.
Sci., vol. 74, No. 12, pp. 5463-5467..
|
Primary Examiner: Hannaher; Constantine
Attorney, Agent or Firm: Flehr, Hohbach, Test, Albritton
& Herbert
Government Interests
This invention was made with United States Government support under
Department of Energy Grant No. DE-FG03-88ER60706 and National
Science Foundation Grant No. BBS 8720382. The Government has
certain rights in this invention.
Parent Case Text
This is a continuation-in-part of co-pending application Ser. No.
463,757 filed Jan. 12, 1990.
Claims
We claim:
1. An improved gel scanner comprising
means for supporting a gel to be scanned,
means for forming a light beam of predetermined wavelength;
a dichroic beam splitter for receiving and directing said beam
toward a supported gel to be scanned;
an objective lens for receiving said beam and focusing the beam in
a selected volume of gel to cause fluorescence emission of light at
a different wavelength and collecting the emitted light from
samples in said selected volume and directing the emitted light to
said dichroic beam splitter which passes said emitted light of
different wavelengths and reflects light at said predetermined
wavelength;
a spatial filter for receiving and passing the emitted light from
said volume while rejecting background and scattered light;
means for providing relative movement between said focused beam and
the gel being scanned,
means for detecting said passed, emitted light and providing output
signals, and
means for processing said signals to provide an image of the
fluorescence from the gel.
2. The method of detecting fluorescence from DNA fragments on a gel
which comprises
exciting a predetermined volume of said gel with light energy of
predetermined wavelength focused therein by an objective lens to
cause said fragments to fluoresce at a different wavelength;
collecting the fluorescently emitted light from said predetermined
volume with said objective lens;
spectrally and spatially filtering said fluorescently emitted light
energy of different wavelength to reject light at said
predetermined and other wavelengths; and
applying the filtered light energy to a detector to generate an
output signal representative of the fluorescence from said
fragments.
3. The method of claim 2 in which the excited volume is scanned
across the gel to obtain emission from adjacent volumes to provide
an output signal for each of said volumes, and processing the
output signals to provide an image of the fluorescence from said
fragments.
4. A scanner for scanning a gel to detect fluorescence from
fluorescence labeled separated sample components comprising
means for supporting a gel to be scanned,
means for applying excitation light at a predetermined wavelength
to a volume in the gel to cause labeled, separated sample
components to fluoresce at a different wavelength, and for
collecting the fluorescence emitted light from said sample at said
different wavelength from said volume and reject the first
wavelength;
means for moving said excitation light and gel relative to one
another to scan the gel;
at least one spectral filter for receiving the collected emitted
light and passing light at said different wavelength while
rejecting light at said predetermined wavelength;
a spatial filter for receiving the emitted light and passing light
from said sample volume while rejecting background and scattered
light;
detecting means for receiving said collective fluorescently emitted
light passed by said spectral and spatial filters and providing
output signals, and
means for receiving and processing said signals to provide an image
of the labeled separated sample components.
5. A scanner as in claim 4 including a laser for supplying said
excitation light.
6. An improved scanner as in claim 5 including a spatial filter for
receiving said light beam and transmitting light to said dichroic
beam splitter.
7. An improved scanner for detecting fluorescence from a
fluorescence labeled sample material carried by a sample carrier
comprising
means for forming a light beam having a predetermined
wavelength;
a dichroic beam splitter for receiving said light beam and
directing it towards an objective lens which receives said beam and
focuses the beam at a spot on the sample carrier whereby to cause
the sample material to fluoresce at a selected volume and for
collecting the fluorescently emitted light from said selected
volume and directing the emitted light to said dichroic beam
splitter, said fluorescent light being at a second wavelength and
said dichroic beam splitter serving to pass the second
wavelength;
a spatial filter for receiving light at said second wavelength and
passing light emitted from said selected volume;
a spectral filter for further filtering said light and passing
light at said second wavelength;
means for moving the light beam and sample carrier relative to one
another to scan the sample carrier,
means for detecting the passed, emitted light of said second
wavelength and providing output signals representative of the
intensity of said detected light, and
means for receiving and processing said signals to provide an image
of the fluorescence of said samples.
8. An improved fluorescence scanner as in claim 7 in which the
carrier is a gel and the sample is a nucleic acid or derivative
thereof.
9. An improved gel scanner comprising
means for supporting a gel to be scanned,
means for forming a light beam of predetermined wavelength;
a dichroic beam splitter for receiving and transmitting said beam
toward a supported gel to be scanned;
an objective lens for receiving said beam and focusing the beam in
a selected volume of gel to cause fluorescence emission of light at
a different wavelength and collecting the emitted light from
samples in said selected volume and directing the emitted light to
said dichroic beam splitter which reflects said emitted light of
different wavelengths and rejects light at said predetermined
wavelength;
a spatial filter for receiving and passing the emitted light from
said volume while rejecting background and scattered light;
means for providing relative movement between said focused beam and
the gel to be scanned,
means for detecting said passed, emitted light and providing output
signals, and
means for processing said signals to provide an image of the
fluorescence from the gel.
Description
BRIEF DESCRIPTION OF THE INVENTION
This invention relates generally to a fluorescence scanner and more
particularly to a laser excited fluorescence gel scanner employing
a confocal microscopic detection system.
BACKGROUND OF THE INVENTION
There is a great deal of interest in the development of automated
DNA mapping and sequencing methodologies which is particularly
important, in view of the recent interest in sequencing the human
genome. The successful completion of this ambitious project will
require improved automation. Sequence data are presently being
added to data banks at a rate of 10.sup.6 bases/year but the human
genome contains 3.times.10.sup.9 base pairs.
The detection system presently used by most workers in DNA
sequencing or mapping involves using radioisotope labeled DNA. The
radioactive slab gels in which the DNA fragments have been
separated are placed against an x-ray film for overnight exposure
of the film. After the exposure and development of the x-ray film,
the sequence or size of the DNA separated fragments are read
directly from the images on the film.
The autoradiographic detection method described above is not only
slow but also requires handling and disposal of hazardous
radioactive materials. The reason autoradiography is still so
widely used is because it uniquely provides the necessary
sensitivity.
There has been great interest in automating the sequence
determination procedures using recent advances in optical,
electronic and computer technology. Autoradiographic films can now
be digitized by a scanning transmission densitometer or video
cameras and the digitized images can be computer processed to
determine DNA sequences. These digitizing and automated sequence
determination systems use autoradiography as the primary detection
method.
In 1986, L. M. Smith, J. Z. Sanders, R. J. Kaiser, P. Hughes, C.
Dodd, C. R. Connell, C. Heiner, S. B. H. Kent and L. E. Hood,
Nature, vol. 321, pp. 674-679, developed a method for detecting
fluorescently labeled DNA on gels which they believe is capable of
sequencing .about.15,000 base pairs per day. They state that one of
the three areas needing development is "increasing the detection
sensitivity of the system thereby allowing less material to be used
per reaction which in turn allows the use of thinner gels having
higher resolution." An apparatus developed by W. Ansorge, A.
Rosenthal, B. Sproat, C. Schwager, J. Stegemann, and H. Voss, Nuc.
Acids Res., vol. 16, pp. 2203-2207 (1988), using a slightly
different protocol, was able to sequence 500 base pairs in 5 hours
with a sensitivity per band of 10.sup.-18 mole or 6.times.10.sup.5
molecules. An analogous approach with similar capabilities was
developed by J. M. Prober, G. L. Trainor, R. J. Dam, F. W. Hobbs,
C. W. Robertson, R. J. Zagursky, A. J. Cocuzza, M. A. Jensen and K.
Baumeister, Science, vol. 238, pp. 336-341 (1987).
The development of a high sensitivity detection system would
obviously be very important. If very small amounts of fluorescence
labeled DNA can be detected on gels, then less labeled DNA is
required and the thickness of the gel can be reduced. A thinner gel
will have higher resolution so it will not have to be run out as
far to resolve the bands. This could result in a major saving in
time. Also, the available fluorescence DNA sequencing systems
require a detection system that is dedicated to the electrophoresis
system during the entire approximate 10 hour run. The detection
system would be more efficiently used if it detected the gels
off-line from the electrophoresis. This could also result in a
major saving of time and increase in throughput.
OBJECTS AND SUMMARY OF THE INVENTION
It is an object of this invention to provide a high sensitivity
fluorescence detection system.
It is another object of the invention to provide a detection system
with improved light collection.
It is another object of the invention to detect fluorescence
emission from nucleic acids, proteins, etc., while rejecting
unwanted background emission.
It is another object of the invention to choose the intensity of
excitation according to the absorption coefficients and emission
life time of the fluorescent molecule to give the optimum
fluorescence emission with minimal background emission.
It is a further object of the invention to choose the illumination
time so that it is less than the photodestruction time of the
fluorescent probe to achieve a high signal-to-noise ratio.
The foregoing and other objects of the invention are achieved by a
fluorescence scanner including means for applying excitation light
to a medium which carries a fluorescently labeled sample to cause
fluorescently labeled sample such as nucleic acids, proteins, etc.
to fluoresce and for collecting the fluorescent emission from said
labeled sample at a selected volume of said medium while rejecting
background and scattered light through confocal spatial filtering
of the detected image.
BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawings, which are incorporated in and form a
part of this specification, illustrate embodiments of the invention
and, together with the description, serve to explain the principles
of the invention:
FIG. 1 shows a laser excited fluorescence gel confocal microscope
scanner in accordance with one embodiment of the invention.
FIG. 2 is a schematic enlarged view of an objective lens assembly,
the gel, gel support, gel objective interface and the volume from
which fluorescence is gathered.
FIG. 3 is a schematic enlarged view of another assembly of the type
shown in FIG. 2.
FIG. 4 is a block diagram of a suitable data acquisition
circuit.
FIG. 5 is a block diagram of another data acquisition circuit.
FIG. 6 shows a typical image formed by the gel scanner of the
present invention.
FIG. 7 shows another laser excited fluorescence gel scanner in
accordance with the present invention.
FIG. 8 shows still another embodiment of a fluorescence gel scanner
in accordance with another embodiment of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
Reference will now be made in detail to the preferred embodiments
of the invention, examples of which are illustrated in the
accompanying drawings. While the invention will be described in
conjunction with the preferred embodiments, it will be understood
that they are not intended to limit the invention to those
embodiments. On the contrary, the invention is intended to cover
alternatives, modifications and equivalents, which may be included
within the invention as defined by the appended claims.
Increasing the fluorescence emission rate from a sample above that
from the background depends on the ability to focus the excitation
light to a very small spot or volume, and to gather the light from
the spot or volume while rejecting background and scattered light.
In fluorescence spectroscopy, it is difficult to illuminate a large
area uniformly or to focus the light to a small spot with
conventional light sources such as arc lamps. When mapping nucleic
acids or their derivatives they are either separated by
polyacrylamide or agarose gel or other gel like separation matrices
with thickness from 100 .mu.m to several millimeters. To detect
nucleic acids in the gel directly by fluorescence emission, one
must get rid of or reduce the scattered light from the surface of
the gel, scattered light from the substrates, and background
emission from the substrates.
In the present invention, a confocal microscope forms an
illuminated volume in the gel. The light comprises a polarized
laser beam oriented so that the background scattering can be
minimized as much as possible by the polarization characteristics
of the scattered light. An oil immersion objective may be used to
match the refractive index of the gel and the objective lens of the
optical microscope so as to reduce the scattering. It is to be
understood that an air gapped objective lens may also be employed.
A confocal optical arrangement is used to reject stray scattering
and emission from unwanted regions of the gel or sample carrier.
Dichroic beam splitters may be used in the optical system to reject
the scattered light by its spectral characteristics. A spatial
filter or beam stop can be used to filter out background and
scattered light.
Referring to FIG. 1, a laser excited confocal microscope scanner is
illustrated. A laser (not shown) is used as the excitation source.
The laser beam 11 is first collimated and focused onto an spatial
filter 12 by an 160.about.mm lens (not shown). After passing
through spatial filter 12, the laser beam is reflected by dichroic
beam splitter 13 through a 100.times., N.A. 1.3 oil immersion
objective 14 such as a Rolyn Optics 80.3610 10.times., NA 1.3. The
dichroic beam splitter reflects light at the wavelength of the
excitation laser beam, but transmits fluorescence light, which is
Stoke's shifted to longer wavelengths by the fluorescent sample.
The objective 14 serves both as focusing lens and collection lens
with very high collection efficiency. The position of the probe,
volume 15, in the gel 16 supported by plate 17, FIG. 2, can be
scanned either by translating the objective and the spatial filter
18 or by translating the sample holder as shown by the arrows 19.
The depth of the probe column is adjusted by moving the same
elements up and down. The fluorescence emission with Stoke's
shifted wavelength is collected by the objective 14 and passed
through the dichroic beam splitter 13 which passes Stoke's shifted
emission. This reduces scattering because it will not pass
scattered light at the laser wavelength.
The light is reflected to spatial filter or image stop 18 by a
second dichroic beam splitter 20. The spatial filter 18 spatially
filters the emitted light and passes it to the phototube detector
21 while rejecting stray, background and scattered light from the
various surfaces. The second dichroic beam splitter 20 reflects
only the Stoke's shifted fluorescence and lets the scattered light
pass through thus providing a second rejection of the background
light. It also rejects scattered or reflected light because its
polarization direction is such as to pass the reflected and
scattered polarized light. The emitted light is detected by a
phototransducer such as a phototube 21.
The use of 160 mm focusing lens, illumination stop or spatial
filter 12, and image stop or spatial filter 18 at 160 mm away from
the objective lens gives this detection system a confocal
arrangement which allows depth profiling of the gel. The depth of
view of the detection system with 100.times. objective is estimated
to be on the order of micrometers. By using this confocal
arrangement, one can selectively probe a DNA sample volume 15 in
the gel. The scattering from the surfaces of the gel is rejected by
the image stop or pinhole 18. Also, the scattering and fluorescence
background from the substrates will be rejected by the spatial
filter. The fluorescence photons can be either directly detected by
the transducer 21 or be passed through a bandpass filter 22 to
further reject background emissions before detection by the
detector 21.
The sample is preferably separated by electrophoresis on a slab
gel. For an immersion objective the slab gel is covered with
nonfluorescent immersion oil 23, FIG. 2 right after electrophoresis
to prevent it from drying and cracking. The gel is then placed on a
computer controlled DC servo motor driven XY translation stage 24
to translate or scan the sample gel past the focused laser volume
15.
FIG. 3 shows a non-immersion objective lens assembly in which the
gel is covered by a thin glass plate 25 which prevents drying out
of the gel.
A microcomputer with an analog-to-digital board or a frame grabber
board is used to digitize data from the data acquisition circuit.
The data acquisition circuit 24, FIG. 4 consists of a preamplifier
26 to amplify signal from photomultiplier tube, a discriminator 27
to reject thermal noises and other low amplitude electronic noises
introduced by the photomultiplier and the preamplifier. A frequency
to voltage converter 28 converts counts to analog signal for
processing with analog-to-digital converter 29. This photon
counting and analog signal conversion method has the fast and
relatively easy processing advantage of analog circuits while it
also has the digital photon counting sensitivity that DC
measurements lack. A more inexpensive version would simply use a
photomultiplier tube, preamplifier and analog-to-digital converter
as in FIG. 5.
The data can also be processed digitally, in which mode, the counts
from the photodetector 21 are processed by a timer/counter 31, FIG.
1, or frequency counter or multichannel scaler directly without the
frequency to voltage converter and the digital-to-analog converter.
Data acquisition circuit 32 provides data to a computer 33. The
computer controls the XY translation stage 24 and displays acquired
image in real time.
FIG. 6 shows an example of a DNA sequencing gel image obtained by
this invention. Each band in the image represents one DNA fragment
of a particular length. The averaged quantity per band in this
image is about 10.sup.5 molecules. The fluorescence images are
contrast-stretched with a histogram equalization method to enhance
the images. The direct imaging of DNA or RNA in the gel by
fluorescence emission has much better linearity than densitometric
scanning of autoradiographic films which usually has logarithmic
response.
FIGS. 7 and 8 show other embodiments of fluorescence gel scanners
incorporating the present invention. Reference numbers have been
applied to parts previously described. In each instance a
collimated laser beam is directly applied through the lens assembly
to a small volume of the gel. The fluorescence from the sample at
the small volume is collected by the objective and focused through
a spatial filter by a lens (40) to a detector (photomultiplier
tube). In other respects, the scanner operates as described above.
The location of the sensed volume within the gel may be selected by
moving the gel, or by moving the lens assembly and spatial
filter.
It is important to point out that these configurations are
fundamentally better than an imaging charge coupled device (CCD) or
charge injection device (CID) coupled to a fluorescence microscope.
A fluorescence microscope is designed to image a very tiny area;
however, the entire field is illuminated so that the incident
intensity in any given small area is low. Furthermore, to image a
large area like a electrophoresis slab gel, one has to use a lens
that can project the whole image onto the camera at one time. This
means using a large F-number lens with low collection efficiency.
Despite the high quantum efficiency of charge coupled devices, the
detection system as a whole is not as sensitive as a photon
counting photomultiplier tube in this kind of application.
It is also worth pointing out that this invention has better
sensitivity over the fluorescence detection system described by
Smith et al., Nature, (1986) simply due to the better collection
efficiency offered by the microscope objective lens and the
background rejection methods intrinsic to the confocal spatial
filtering of the detected light.
In a preliminary experiment the detection limits of this invention
for DNA sequencing gel reached about 1.times.10.sup.5 molecules per
band on the average with a 250 .mu.m thick gel and 3 mm wide sample
well. Although the on-line detection method described by Smith, et
al. (1986) has advantages, the off-line detection method taught in
this invention doesn't require a laser and detection system
dedicated to every electrophoresis system. This allows several gels
being run simultaneously to have higher throughput and is more
economical.
In summary, the laser excited confocal fluorescence scanning system
taught herein is a very sensitive method for detecting DNA and RNA
in gels. This invention employs a confocal microscope with a high
numerical aperture objective to achieve the highest collection
efficiency possible. The invention teaches polarization, spectral
filtering, spatial filtering and refractive index matching
principles to effectively reduce background in order to obtain
optimum detection limits. The electronic gating of noises by photon
counting with frequency to voltage conversion provides further
improvement in signal-to-noise. The translation of the gel across
the laser beam provides a means to image a large electrophoresis
gel with high sensitivity and spatial resolution. The ultimate
spatial resolution in this fluorescence imaging system is
determined by the spot size of the laser beam which can be as small
as 1-2 .mu.m in diameter using the optical elements described in
this invention. The combination of high spatial resolution and low
detection limits of this fluorescence imaging system means that
very low detection limits can be easily achieved which approach the
limits of autoradiography.
As an improved instrument for fluorescence detection and imaging of
electrophoresis gels, this invention should be of interest to
biological or biochemical researchers, particularly those working
in DNA sequencing and mapping. This invention can detect DNA
samples that cannot be detected by conventional fluorescence
detection methods, and it provides a faster and safer way to do DNA
sequencing without handling radioactive materials. It is apparent
that the invention may be used to detect and image fluorescent
labeled molecules, proteins, virus and bacteria, etc., which are
electrophoretically or otherwise separated on a variety of carriers
such as membranes, filter paper, petrie dishes, glass substrates,
etc.
The foregoing descriptions of specific embodiments of this
invention have been presented for purposes of illustration and
description. They are not intended to be exhaustive or to limit the
invention to the precise forms disclosed, and many modifications
and variations are possible in light of the above teaching. The
embodiments were chosen and described in order to best explain the
principles of the invention and its practical application, to
thereby enable others skilled in the art to best use the invention
and various embodiments with various modifications as are suited to
the particular use contemplated. It is intended that the scope of
the invention be defined by the claims appended hereto and their
equivalents.
* * * * *