Note: Descriptions are shown in the official language in which they were submitted.
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SIMULTANEOUS IMAGE ACQUISITION USING MULTIPLE
FLUOROPHORE PROBE DYES
BACKGROUND OF THE INVENTION
The subject invention relates generally to an improved scanner of the type
that
scans specimens for performing subsequent computer analysis on the specimens.
Micro array biochips are presently being used by several biotechnology
companies
for scanning genetic DNA samples applied to biochips into computerized images.
These
chips have small substrates with thousands of DNA samples that represent the
genetic
codes of a variety of living organisms including human, plant, animal, and
pathogens.
They provide researchers with information regarding the DNA properties of
these
organisms. Experiments can be conducted with significantly higher throughput
than
previous technologies by using these biochips. Biochip technology is used for
genetic
expression, DNA sequencing of genes, food and water testing for harmful
pathogens, and
diagnostic screening. Biochips may be used in pharmacogenomics and proteomics
research aimed at high throughput screening for drug discovery.
DNA samples are extracted from a sample and are tagged with a fluorescent dye
having a molecule that, when excited by a laser, will emit light of various
colors. Often,
a DNA sample is tagged with multiple dyes. Each of these dyes is utilized to
illuminate
different characteristics of a particular DNA sample. These fluorescently
tagged DNA
samples are then spread over the chip. A DNA sample will bind to its
complementary
(cDNA) sample at a given array location. A typical biochip is printed with a
two
dimensional array of thousands of cDNA samples, each one unique to a specific
gene.
Once the biochip is printed, it represents thousands of specimens in an area
usually
smaller than a postage stamp.
A microscope collects data through a scanning lens by scanning one pixel of a
specimen at a time. The scanning lens projects emitted light from the specimen
onto a
sensor that is manipulated along a predetermined pattern across the chip
scanning an
entire biochip one pixel at a time. The pixels are relayed to a controller
that sequentially
connects the pixels to form a complete, computerized biochip image. The
fluorescent
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dyes that are suitable for use in this capacity have spectral arrays that
overlap when
excited. The overlapping of the spectral arrays can skew the scanning results
and can
lead to inaccurate computer analysis of the DNA samples being scanned.
It would be desirable to perform scanning of DNA samples tagged with multiple
dyes and yet prevent the overlap of the spectral arrays from adversely
affecting data
generated. Therefore, a need exists for an optical instrument capable of
filtering the
overlapping portions of the spectral arrays from multiple dyes while
performing high
speed scanning of current practice.
SUMMARY OF THE INVENTION AND ADVANTAGES
The present invention provides an optical instrument assembly that scans a DNA
specimen one pixel at a time and relays the scan to a controller that connects
the pixels
forming a computerized biochip image of the specimen. The assembly includes a
transmitter for emitting an optical signal having at least a first and a
second spectral array.
A reflector directs the optical signal onto the specimen, which is treated
with fluorescent
dyes that are excited by the various spectral arrays in the optical signal. A
detector
includes an objective lens that focuses the emitted optical signal from the
specimen onto
a sensor. The sensor transmits the emitted optical signal to a controller one
pixel at a
time.
A first drive mechanism varies the position of the optical signal transmitted
onto
the specimen in a forward and reverse direction. A second drive mechanism
varies the
position of the specimen relative to the optical signal. In this manner, a
complete scan
of the specimen is performed and transmitted to a controller one pixel at a
time.
The controller terminates detection of one of the spectral arrays while
varying the
position of the optical signal in the forward direction and terminates
detection of the other
spectral array while varying the position of the optical signal in the reverse
direction. By
detecting only one spectral array at a time, the problem of overlapping
spectral arrays
from multiple dyes is eliminated improving the accuracy of the computer
analysis
performed upon the DNA sample.
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BRIEF DESCRIPTION OF THE DRAWINGS
Other advantages of the present invention will be readily appreciated as the
same
becomes better understood by reference to the following detailed description
when
considered in connection with the accompanying drawings wherein:
Figure 1 is a detailed view of an optical instrument of the present invention;
Figure 2 is a plan view of a biochip specimen of the present invention showing
the movement of the scanning objective lens;
Figure 3 is a side view of the first drive mechanism;
Figure 4 is a top view of the second drive mechanism.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
The optical instrument assembly of the present invention is generally shown in
Figure 1 at 10. The assembly includes a transmitter 12 for emitting an optical
signal 14.
In the preferred embodiment, the transmitter 12 comprises a laser. Figure 1
shows three
transmitters 12a-c, each emitting an optical signal 14a-c having a different
spectral array.
Additional transmitters 12 may be introduced to the assembly 10 as needed.
A reflector 30 directs the optical signal 14 onto a specimen 90. The reflector
30
includes a plurality of turn mirrors 32. Figure 1 shows three turn minors 32a-
c
corresponding to the same number of transmitters 12a-c. Each optical signal
14a-c is
reflected by the turn mirrors 32a-c into corresponding beam combiners 34a-c.
The beam
combiners 34a-c, known as dichroic filters, transmit light of one wavelength
while
blocking other wavelengths. The beam combiners 34a-c collect the individual
optical
signals 14a-c into a combined beam along a single path and direct the beam
towards a
beam splitting mirror 20. The beam splitting minor 20 includes an opening 22
through
which the combined optical signals 14a-c travel. Subsequently, the combined
optical
signals 14a-c reflect off a ninety degree fold mirror 36 located immediately
above a
scanning objective lens 52, which focuses the combined optical signals 14a-c
onto a
section of the specimen 90. A first drive mechanism 50 varies the position of
the
combined optical signal 14a-c onto the specimen 90 as will be explained
further
hereinbelow.
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The specimen 90 is treated with a plurality of dyes having fluorescent
properties
when subjected to the optical signal 14a-c. The specimen 90, having been
treated with
the dyes, and illuminated with the optical signal 14, emits the optical signal
44 at a
spectral array corresponding to the dye selected. Different dyes may be used
to examine
S different specimen 90 properties. Multiple dyes may be used to examine
different
properties of the same specimen 90 simultaneously. Typically, at least a first
dye and a
second dye will be used. The first dye is chosen to be illuminated with
optical signal 14a
and emits optical signal 44a having a first spectral array, and the second dye
is chosen to
be illuminated with optical signal 14b and emits optical signal 44b having a
second
spectral array.
The assembly 10 includes a detector 40 with a sensor 42 for detecting a
emitted
optical signal 44 from the specimen 90. The emitted optical signal 44 reflects
off the
opposite side of the beam splitting mirror 20 through a plurality of beam
splitters 38a-b
to separate the emitted optical signal 44 into individual signals 44a-c
corresponding to
different spectral arrays from the various dyes. Each individual signal passes
though an
emission filter 46a-c and is focused by a detector lens 48a-c into a pinhole.
The
individual signals 44a-c proceed through the pinholes to contact individual
sensors 42a-c.
The sensors 42a-c are in communication with a controller 80 as will described
in further
detail hereinbelow.
As shown in Figure 2, the objective lens 52 is moved in forward and reverse
directions along the x-axis of the specimen 90 collecting data in each
direction. The
specimen 90 does not move in the x direction. The specimen 90 is moved in the
y
direction incrementally each time a scan is about to be started in the x
direction. In this
manner, a rectangular zigzag scanning pattern is performed upon the specimen
90.
Figure 3 shows a first drive mechanism 50 that varies the position of the
combined optical signal 14a-c on the specimen 90 in a forward and reverse
direction.
The first drive mechanism 50 preferably employs a galvanometric torque motor
54 to
rotate a sector-shaped cam 56 over an angle between plus forty degrees and
negative forty
degrees. The circular portion of the cam 56 is connected to the carnage 58 via
a set of
roll-up, roll-off thin, high strength steel wires 66a-b. The scanning
objective lens 52 is
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attached to the carnage 54. The radius of the cam 56 is such that its rotation
will cause
the carnage 58 to travel a linear distance along a rail 60 commensurate with
the length
of the scan along the x-axis.
The controller 80 communicates with the transmitters 12a-c and the sensors 42a-
c.
S The sensors 42a-c relay to the controller 80 the emitted spectral arrays
from the specimen
90 for the controller to reconstruct the computerized image of the DNA sample.
The
controller 80 is formatted to modify the scanning pattern to prevent the
detection of
overlapping spectral arrays, which would otherwise produce inaccurate
computerized
image of the DNA sample. When the first drive mechanism SO drives the combined
optical signal 14a-c in the forward direction, information from the first dye
will be
acquired. When the first drive mechanism 50 drives the combined optical signal
14a-c
in the rearward direction, information from the second dye will be acquired.
To exclude information from the second dye, the controller 80 will deactivate
either the sensor 42b that reads the second dye, or the transmitter 12b that
excites the
fluorescent properties of the second dye. Likewise, to exclude information
from the first
dye, the controller will deactivate either the sensor 42a that reads the first
dye, or the
transmitter 12a that excites the fluorescent properties of the first dye.
In order to produce an accurate computerized DNA image, the controller 80 must
correlate the forward and rearward scans. In order to calculate an accurate
correlation,
the distance between consecutive scan lines should be no more than forty
percent of the
height of the optical resolution of the optical system utilized by the
assembly 10.
Figure 4 shows a second drive mechanism 70 employing a stepper motor 72 to
drive a precision screw 74 in a known manner. A nut 76 on the screw 74 is
attached to
the carnage 58 so that any rotation of the screw 74 will cause the carriage 58
to move
along a linear rail 60. The carriage in turn is equipped with a tray 76 which
includes
retainers 78 to hold a specimen 90 slide in a position and orientation that is
repeatable
within an accuracy required by optical focus and alignment criteria. The rail
60 and the
stepper motor 72 are attached to the frame of the second drive mechanism 70.
The first and second drive mechanisms 50, 70 transmit location information to
the
controller 80. The controller 80 uses the location information to map the scan
data
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received from the sensors 42a-c. A scanning accuracy of one micron is required
to
accurately map the scan using data from both directions scanned on the x-axis.
The invention has been described in an illustrative manner, and it is to be
understood that the terminology which has been used is intended to be in the
nature of
S words of description rather than of limitation.
Obviously, many modifications and variations of the present invention are
possible in light of the above teachings. It is, therefore, to be understood
that within the
scope of the appended claims, wherein reference numerals are merely for
convenience
and are not to be in any way limiting, the invention may be practiced
otherwise than as
specifically described.
