U.S. patent application number 16/742783 was filed with the patent office on 2020-12-17 for high speed scanning system with acceleration tracking.
The applicant listed for this patent is Apton Biosystems, Inc.. Invention is credited to Robert HARTLAGE, Paul HEILMAN, Windsor OWENS, Bryan P. STAKER, David STERN, Edvinas ZIZMINSKAS.
Application Number | 20200393691 16/742783 |
Document ID | / |
Family ID | 1000005051476 |
Filed Date | 2020-12-17 |
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United States Patent
Application |
20200393691 |
Kind Code |
A1 |
OWENS; Windsor ; et
al. |
December 17, 2020 |
HIGH SPEED SCANNING SYSTEM WITH ACCELERATION TRACKING
Abstract
Disclosed herein is a high throughput optical scanning device
and methods of use. The optical scanning device and methods of use
provided herein can allow high throughput scanning of a
continuously moving object with a high resolution despite
fluctuations in stage velocity. This can aid in high throughput
scanning of a substrate, such as a biological chip comprising
fluorophores. Also provided herein are improved optical relay
systems and scanning optics.
Inventors: |
OWENS; Windsor; (San
Francisco, CA) ; STAKER; Bryan P.; (San Ramon,
CA) ; HARTLAGE; Robert; (Sunnyvale, CA) ;
ZIZMINSKAS; Edvinas; (Dublin, CA) ; STERN; David;
(Mountain View, CA) ; HEILMAN; Paul; (San Ramon,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Apton Biosystems, Inc. |
Pleasanton |
CA |
US |
|
|
Family ID: |
1000005051476 |
Appl. No.: |
16/742783 |
Filed: |
January 14, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15910778 |
Mar 2, 2018 |
10585296 |
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16742783 |
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62467048 |
Mar 3, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
H04N 5/232 20130101;
G02B 26/101 20130101; H04N 3/08 20130101; H04N 5/2328 20130101;
G01D 5/34746 20130101; G02B 27/644 20130101 |
International
Class: |
G02B 27/64 20060101
G02B027/64; H04N 5/232 20060101 H04N005/232; G01D 5/347 20060101
G01D005/347; G02B 26/10 20060101 G02B026/10; H04N 3/08 20060101
H04N003/08 |
Claims
1.-67. (canceled)
68. An optical scanning system for imaging a moving substrate,
comprising: a. a stage configured to: i. undergo movement along an
axis, and ii. support a substrate comprising a plurality of fields
during said movement along said axis; b. a camera in optical
communication, through an optical path, with a field of said
plurality of fields, when said substrate is supported by said
stage, and wherein said camera is configured to capture an image of
said substrate comprising said field; c. a velocity tracking mirror
and an acceleration tracking mirror mounted along said optical
path, wherein said velocity tracking mirror and said acceleration
tracking mirror are configured to maintain said optical path to
stabilize said image of said substrate during said movement of said
substrate.
69. The system of claim 68, further comprising a first electrical
motor, and wherein a movement of said velocity tracking mirror is
actuated by said first electrical motor.
70. The system of claim 69, wherein said first electrical motor is
a galvanometer.
71. The system of claim 68, further comprising a second electrical
motor, wherein a movement of said acceleration tracking mirror is
actuated by said second electrical motor.
72. The system of claim 71, wherein said second electrical motor is
a piezoelectric actuator.
73. The system of claim 68, wherein said velocity tracking mirror
and said acceleration tracking mirror are adjacent.
74. The system of claim 68, further comprising an objective lens,
wherein said objective lens is configured to move along said
optical path and wherein a movement of said objective lens is a
function of a movement of said field out of a focal plane of said
objective lens.
75. The system of claim 74, further comprising a third electrical
motor and wherein a movement of said objective lens is actuated by
said third electrical motor.
76. The system of claim 68, wherein a movement of said velocity
tracking mirror is a function of a velocity measurement of said
movement of said stage along said axis.
77. The system of claim 76, wherein said movement of said
acceleration tracking mirror is a function of a change in velocity
measurement of said movement of said stage along said axis.
78. The system of claim 77, wherein said function of said velocity
measurement and said function of said change in velocity
measurement are generated by a linear displacement sensor, wherein
said linear displacement sensor determines a positional measurement
of said stage.
79. The system of claim 78, wherein said linear displacement sensor
is a linear encoder.
80. The system of claim 68, wherein said optical path comprises a
filter configured to reduce a transmission of excitation light to
said camera.
81. The system of claim 80, wherein a light source configured to
generate said optical path is displaced adjacent to said stage and
is not adjacent to said camera.
82. The system of claim 68, further comprising an additional pair
of mirrors comprising a second velocity tracking mirror and a
second acceleration tracking mirror, wherein said additional pair
of mirrors is mounted along said optical path and wherein said
additional pair of mirrors is configured to motion along an
additional axis.
83. A method of imaging a moving substrate, wherein said substrate
comprises one or more fields, the method comprising: a. disposing
said substrate on a stage; b. configuring a camera to be in optical
communication with said one or more fields of said substrate by an
optical path; c. actuating a movement of said stage and configuring
a velocity tracking mirror and an acceleration tracking mirror to
maintain said optical path to stabilize an image of said substrate
during a movement of said stage; and d. concurrent with said
movement, imaging said field passing through an objective lens
using said camera.
84. The method of claim 83, wherein (d) further comprises actuating
a movement of said objective lens along said axis and wherein said
movement of said objective lens is a function of a movement of said
one or more fields out of a focal plane of said objective lens.
85. The method of claim 83, wherein (c) comprises actuating a
movement of said velocity tracking mirror.
86. The method of claim 85, wherein said movement of said velocity
tracking mirror is a function of an anticipated velocity of said
stage.
87. The method of claim 85, wherein said movement of said velocity
tracking mirror is a function of a velocity measurement of said
movement of said stage along said axis.
88. The method of claim 85, wherein (c) comprises actuating a
movement of said acceleration tracking mirror.
89. The method of claim 88, wherein a movement of said acceleration
tracking mirror is a function of a change in velocity measurement
of said movement of said stage along said axis.
90. The method of claim 89, wherein (c) comprises configuring a
linear displacement sensor to generate said function of said
velocity measurement and said function of said change in velocity
measurement of said stage.
91. The method of claim 83, wherein (b) further comprises
displacing a light source configured to generate said optical path
is displaced adjacent to said stage and is not adjacent to said
camera.
92. The method of claim 83, wherein (b) further comprises
displacing a filter along said optical path.
93. The method of claim 83, wherein (c) further comprises actuating
a movement of an additional pair of mirrors, said additional pair
of mirrors comprising a second velocity tracking mirror and a
second acceleration tracking mirror, wherein said additional pair
of mirrors is mounted along said optical path and wherein said
additional pair of mirrors is configured to motion along an
additional axis.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. patent
application Ser. No. 15/910,778, filed on Mar. 2, 2018, which
claims the benefit of U.S. Provisional Application No. 62/467,048,
filed on Mar. 3, 2017, the entire disclosures of which are hereby
incorporated by reference in their entireties.
FIELD OF THE INVENTION
[0002] The invention relates to methods and instruments for
generating a stable image of a continuously moving substrate in an
optical system.
BACKGROUND OF THE INVENTION
[0003] Typical single-molecule, single-fluor sensitivity biological
fluorescent optical scanning systems require low noise cameras with
long exposure times. These systems often require a high precision
and stable imaging platform situated on granite or equivalent. In
addition, these systems employ "step and repeat" staging which
necessitate high acceleration and deceleration as well as high mass
in order to achieve high throughput, stable imaging of multiple
fields. To scan a large area chip (2000 mm2) in a short amount of
time (-5 min) at high magnification requires frame imaging times
shorter than step and repeat systems allow.
[0004] An "image on the fly" approach is needed to prevent a loss
in throughput due to stage accelerations and settling time inherent
to the step and repeat systems. Traditional image on the fly
applications require sample stages that can provide near-constant
(--+/-0.05%) velocity, and scanning optics that image the sample as
it moves. If the stage velocity is not near-constant throughout its
travel, then the scanning optics will not image the exact same
region of the sample as the stage moves. This can result in a
blurry image (e.g., with a pixel smear of--+/-3 pixels). This
problem is typically solved by utilizing expensive stages that
provide near constant velocity by using heavy stages and powerful
stage drives. Unfortunately, this adds to the cost of the product
and makes it impractical to use as a benchtop system.
[0005] Typical low-cost, compact and/or lightweight stages are
built with components that have various surface irregularities such
as pits, burrs, machining grooves, divots and misshapen cavities.
These irregularities usually result in velocity that is not
constant. For instance, a burr or a divot in the ways of a stage
will cause the stage to momentarily slow down and then possibly
speed up before returning to the velocity it had before it
encountered the irregularity. The velocity fluctuations of the
stage make the use of these low-cost, smaller components
incompatible with current image on the fly high throughput scanning
approaches due to the generation of unacceptable levels of image
blur.
[0006] What is needed therefore, are improved scanning optics that
increase the velocity fluctuation tolerance, allowing an image with
increased stability (e.g., reduced pixel smear) to be obtained
using image on the fly scanning with single fluor sensitivity in
smaller, lightweight, and low-cost optical scanning systems.
SUMMARY OF THE INVENTION
[0007] The instant invention is based, at least in part, on the
discovery of new methods and devices to reduce pixel smear in the
imaging of an object on a moving stage.
[0008] Accordingly, provided herein is an optical scanning system
for imaging a moving substrate, comprising a stage, said stage
capable of moving along an axis, said stage configured to hold a
substrate comprising a plurality of fields; an objective lens; a
camera capable of acquiring an image of one of said plurality of
fields through the objective lens, said image acquired via an
optical path defined from one of said plurality of fields through
said objective lens to said camera during acquisition of said
image; a velocity tracking mirror mounted along said optical path;
a first electrical motor operably coupled to said velocity tracking
mirror to adjust the angle of the tracking mirror along said axis
of stage movement in said optical path; a controller module
operably coupled to said first electrical motor to send a first
driving signal to said first electrical motor, wherein said first
driving signal is a function of a velocity measurement of the stage
movement along said axis; an acceleration tracking mirror mounted
along said optical path; a second electrical motor operably coupled
to said acceleration tracking mirror to adjust the angle of the
acceleration tracking mirror along said axis of stage movement in
said optical path, wherein said controller module is operably
coupled to said second electrical motor to send a second driving
signal to said second electrical motor, wherein said second driving
signal is a function of the change of the stage velocity along said
axis.
[0009] In some embodiments, the first or second driving signal is
an electrical signal. In some embodiments, the first driving signal
comprises a non-sinusoidal waveform. In some embodiments, the
non-sinusoidal waveform is a sawtooth wave. In some embodiments,
the first electrical motor is a galvanometer. In some embodiments,
the second electrical motor is a piezoelectric actuator. In some
embodiments, the first electrical motor or said second electrical
motor are dual axes motors.
[0010] In some embodiments, the system further comprises a linear
displacement sensor operably coupled to said controller module to
send a signal comprising a positional measurement of said substrate
or said stage to said controller module. In some embodiments, the
linear displacement sensor is a linear encoder.
[0011] In some embodiments, the first driving signal is a function
of a velocity determined from said positional measurement. In some
embodiments, the second driving signal is a function of a change in
velocity determined from said positional measurement.
[0012] In some embodiments, the first or second signal comprises a
waveform that is a function of the field scan frequency. In some
embodiments, the first or second signal comprises a waveform that
is a function of the imaging duty cycle.
[0013] In some embodiments, the movement of said velocity tracking
mirror and said acceleration tracking mirror reduce a tracking
error of said field by said camera as compared to without movement
of said acceleration tracking mirror. In some embodiments, the
tracking error is reduced to less than 0.1%. In some embodiments,
the tracking error is reduced to less than 1 pixel.
[0014] In some embodiments, the velocity tracking mirror and the
acceleration tracking mirror are adjacent components along said
light path.
[0015] In some embodiments, the system comprises a plurality of
cameras. In some embodiments, the system further comprises a beam
splitter mounted along said light path, wherein said beam splitter
is mounted along said light path after said velocity tracking
mirror and acceleration tracking mirror and before said plurality
of cameras.
[0016] In some embodiments, the system further comprises an
illumination path, said illumination path extending from an
illumination element to one of said plurality of fields.
[0017] In some embodiments, the illumination element comprises an
excitation laser operably mounted to transmit an excitation light
to said field, wherein said optical path comprises fluorescent
light emitted from said field to said camera. In some embodiments,
the excitation light is not transmitted to said camera. In some
embodiments, the illumination element comprises an illumination
light operably mounted to transmit an illumination light to said
field. In some embodiments, the illumination light is mounted
underneath the field such that the optical path comprises light
transmitted through said field and to said camera. In some
embodiments, the illumination light is mounted above or transverse
to said field such that the optical path comprises light reflected
by said field and to said camera.
[0018] In some embodiments, the system further comprises a third
electrical motor operably mounted to the objective lens to move the
objective lens along said optical path, thereby maintaining said
field in focus. In some embodiments, the third electrical motor is
operably connected to said controller module to receive a third
driving signal that is a function of the movement of the field out
of the focal plane, such that the objective lens is moved to
maintain said field in focus by said camera.
[0019] In some embodiments, the system further comprises at least
one additional pair of mirrors comprising a second velocity
tracking mirror and a second acceleration tracking mirror, wherein
said mirrors are operably mounted to said device to reduce a
tracking error of said field by said camera along a different
axis.
[0020] Also provided herein is a method of imaging a plurality of
fields on a moving substrate, comprising providing an optical
scanning system comprising a moveable stage holding a substrate
comprising a plurality of fields, a camera, an objective lens, a
velocity tracking mirror, and an acceleration tracking mirror;
moving said moveable stage along an axis, thereby moving said
substrate comprising a plurality of fields along said axis; and
concurrent with said movement, capturing an image of one of said
plurality of fields passing through said objective lens using said
camera, wherein said image of said field is stabilized during said
image capture by rotating said velocity tracking mirror as a
function of a velocity of the moveable stage along said axis, and
rotating said acceleration tracking mirror as a function of a
change in the velocity of the moveable stage along said axis.
[0021] In some embodiments, the method of imaging a plurality of
fields on a moving substrate further comprises obtaining a
measurement of the velocity of said moveable stage, said substrate,
or said field along said axis, and adjusting said first driving
signal as a function of said velocity. In some embodiments, the
method of imaging a plurality of fields on a moving substrate
further comprises determining a change in velocity of said moveable
stage from a plurality of velocity measurements, and adjusting said
second driving signal as a function of said change in velocity.
[0022] In some embodiments, the rotation of the velocity tracking
mirror or the acceleration tracking mirror is performed based on
said measured velocity or said measured change in velocity. In some
embodiments, the first driving signal is a function of an
anticipated velocity of said stage. In some embodiments, the second
driving signal is a function of an anticipated change in velocity
of said stage.
[0023] In some embodiments, the velocity tracking mirror is
operably coupled to a first electric motor. In some embodiments,
the first electric motor is a galvanometer. In some embodiments,
the optical scanning system comprises a controller module, and
wherein said first electric motor is operably coupled to said
controller module.
[0024] In some embodiments, rotating said velocity tracking mirror
comprises sending a first driving signal from said controller
module to said first electric motor. In some embodiments, the first
driving signal is a function of a measured or predetermined
velocity of the substrate.
[0025] In some embodiments, the acceleration tracking mirror is
operably coupled to a second electric motor. In some embodiments,
the electric motor is a piezoelectric actuator. In some
embodiments, the electric motor is operably coupled to said
controller module. In some embodiments, the acceleration tracking
mirror comprises sending a second driving signal from said
controller module to said second electric motor.
[0026] In some embodiments, the second driving signal is a function
of a measured or predetermined change in the velocity of the
substrate. In some embodiments, the second driving signal is a
function of a deviation of said velocity from the velocity used to
determine said first driving signal.
[0027] In some embodiments, the velocity tracking mirror and the
acceleration tracking mirror are adjacent.
[0028] In some embodiments, the movement of said velocity tracking
mirror and said acceleration tracking mirror reduce a tracking
error of said field by said camera as compared to without movement
of said acceleration tracking mirror. In some embodiments, the
tracking error is reduced to less than 0.1%. In some embodiments,
the tracking error is reduced to less than 1 pixel.
[0029] In some embodiments, the method further comprises adjusting
the location of the objective lens along the optical path to
maintain said field in focus during said image capture. In some
embodiments, the adjustment of the objective lens maintains an
intensity jump between two adjacent pixels of said image of greater
than 50%, 60%, 70%, 80%, or 90%.
[0030] In some embodiments, the optical scanning system further
comprises a second velocity tracking mirror and a second
acceleration tracking mirror, further comprising, concurrent with
said movement of said moveable stage and said image capture of one
of said plurality of fields: rotating said second velocity tracking
mirror as a function of a velocity of the moveable stage along a
second axis, and rotating said second acceleration tracking mirror
as a function of a change in the velocity of the moveable stage
along said second axis thereby stabilizing imaging of said field
for at least two axes simultaneously.
[0031] In some embodiments, the method further comprises rotating
each of a plurality of pairs of velocity tracking and acceleration
tracking mirrors to stabilize an image for a corresponding
plurality of distinct axes.
[0032] In some embodiments, the frequency of image capture is at
least 20 Hz, 40 Hz, 60 Hz, 80 Hz, 100 Hz, 120 Hz, 140 Hz, 160 Hz,
180 Hz, or 200 Hz. In some embodiments, the duty cycle of the image
capture is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%. In
some embodiments, the duty cycle of the image capture is at least
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.
[0033] Also provided herein is a method of reducing positioning
error in an image obtained from a moving stage, comprising:
measuring a velocity of said moving stage; determining an error
correction term from said measured velocity as a function of the
difference between the measured velocity of said stage and an
anticipated velocity of said stage; generating a driving signal as
a function of said error correction term; and sending said driving
signal to an electrical motor, wherein said electrical motor is
operably connected to a tracking mirror to actuate rotation of said
tracking mirror. In some embodiments, the motor is a galvanometer
or a piezoelectric actuator.
[0034] Also provided herein is an optical scanning system for
imaging a moving substrate, comprising: a stage, said stage capable
of moving along an axis, said stage configured to hold a substrate
comprising a plurality of fields; an objective lens; a camera
capable of acquiring an image of one of said plurality of fields
through the objective lens, said image acquired via an optical path
defined from one of said plurality of fields through said objective
lens to said camera during acquisition of said image; a motion
tracking mirror mounted along said light path; an electric motor
operably coupled to said motion tracking mirror to actuate angular
motion of the tracking mirror along said axis of stage movement in
said optical path; and a controller module operably coupled to said
electric motor to send a driving signal to said electric motor,
wherein said controller module is capable of generating said
driving signal as a function of a velocity fluctuation of said
stage or substrate movement along said axis.
[0035] In some embodiments, the device comprises a velocity sensor
in electrical communication with the controller module, said
velocity sensor capable of detecting positional or velocity
information of the substrate or stage and sending said information
to the controller module, wherein said controller module is
configured to generate said driving signal as a function of the
velocity signal received from the velocity sensor.
[0036] In some embodiments, the sensor is a linear encoder. In some
embodiments, the linear encoder is a non-interferometric encoder.
In some embodiments, the linear encoder is optical, magnetic,
capacitive, inductive, or uses an Eddy current. In some
embodiments, the sensor is calibrated for velocity feedback across
the moveable stage.
[0037] In some embodiments, the driving signal is a function of
both a pre-determined velocity and a measured velocity. In some
embodiments, the stage comprises a mechanical bearing positioned to
facilitate movement of said stage along an axis. In some
embodiments, the objective lens has a magnification selected from
the group consisting of: 5.times., 10.times., 20.times., 30.times.,
40.times., 50.times., 60.times., 70.times., 80.times., 90.times.,
or 100.times..
[0038] Also provided herein is a method of imaging a plurality of
fields on a moving substrate, comprising: providing an optical
scanning system comprising a moveable stage holding a substrate
comprising a plurality of fields, an objective lens, a camera, a
motion tracking mirror, and an electric motor operatively coupled
to said motion tracking mirror to effect movement of said motion
tracking mirror to track said movement of said moveable stage along
said axis during an image capture, and to return said motion
tracking mirror to an initial position after said image capture;
moving said moveable stage along an axis, thereby moving said
substrate comprising a plurality of fields along said axis; and
generating an image for each of M fields of said substrate,
performing at least M image capture cycles during movement of said
moveable stage along said axis, each cycle comprising: providing a
cycle M driving signal to an electric motor to control movement of
said tracking mirror to track the velocity of said moveable stage
along said axis; capturing an image of said field while said
tracking mirror is tracking said moving stage; and determining an
average velocity of said field, wherein said average velocity is
used to generate a cycle M+1 driving signal to control movement of
said electric motor during cycle M+1.
[0039] In some embodiments, the frequency of image capture is at
least 20 Hz, 40 Hz, 60 Hz, 80 Hz, 100 Hz, 120 Hz, 140 Hz, 160 Hz,
180 Hz, or 200 Hz. In some embodiments, the duty cycle of the image
capture is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%. In
some embodiments, the duty cycle of image capture is at least 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.
[0040] In some embodiments, the method further comprises performing
an initial cycle to determine an average velocity of said field,
wherein no image capture occurs. In some embodiments, the M+1
driving signal comprises a correction term that is a function of
the difference between a measured velocity and a desired velocity
of said moveable stage along said axis. In some embodiments,
determining said average velocity of said field comprises measuring
one or more positions of a field at a time. In some embodiments,
the average velocity of said field further comprises comparing said
measured position of said field at said time with a previously
measured position and time of another field.
[0041] In some embodiments, the velocity feedback loop duration
from positional measurement of field M to providing said M+1
driving signal is no more than 100 ms and could be as low as 2 ms,
90 ms, 80 ms, 70 ms, 60 ms, 50 ms, 40 ms, or 30 ms. In some
embodiments, the average velocity is determined by collecting
information about the position of the substrate at a frequency of
no more than 250 kHz, 200 kHz, 150 kHz, 100 kHz, 50 kHz, 20 kHz, 10
kHz, 5 kHz, 2 kHz, 1000 Hz, 500 Hz, 240 Hz, 120 Hz, 60 Hz, or 30
Hz.
[0042] In some embodiments, the generated image has a pixel smear
of no more than +/-one pixel. In some embodiments, the pixel
comprises a cross-sectional distance along said axis of about 150
nm on said substrate.
[0043] In some embodiments, the image is generated from a substrate
moving at a velocity in a range from 100 p.m./second to 1,000
mm/second. In some embodiments, the movement of said moveable stage
along said axis comprises velocity fluctuations in the range of
0.1% to 1% of the average velocity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0044] The foregoing and other objects, features and advantages
will be apparent from the following description of particular
embodiments of the invention, as illustrated in the accompanying
drawings in which like reference characters refer to the same parts
throughout the different views. The drawings are not necessarily to
scale, emphasis instead placed upon illustrating the principles of
various embodiments of the invention.
[0045] FIG. 1 is a diagram of components of an optical scanning
device along an optical path from a substrate to a detector
comprising an acceleration tracking mirror and a velocity tracking
mirror (i.e., a dual mirror embodiment) according to an embodiment
of the invention.
[0046] FIG. 2 is a representation of a sawtooth waveform to control
the motion of a moving stage tracking mirror, such as a velocity
tracking mirror, and its correlation to the change in position of a
moveable stage along an axis over time according to an
embodiment.
[0047] FIG. 3 provides an example of waveforms that can be used to
generate driving signals for a velocity tracking mirror and an
acceleration tracking mirror (in a dual mirror embodiment) to
stabilize an image of a moving substrate based on a measured or
anticipated stage velocity error and an anticipated stage
velocity.
[0048] FIG. 4 is a diagram of components of an optical scanning
device along an optical path from a substrate to a detector
comprising a single motion tracking mirror (i.e., a single-mirror
embodiment) according to an embodiment of the invention.
[0049] FIG. 5 provides an example of waveforms that can be used to
generate driving signals for a motion tracking mirror (in a single
mirror embodiment) to stabilize an image of a moving substrate
based on a measured or anticipated stage velocity error and an
anticipated stage velocity.
[0050] FIG. 6 provides a schematic of one possible implementation
of a field level feed-forward mechanism to provide a correction
term to adjust a drive signal provided to a mirror of the device
capable of moving in response to velocity fluctuations of the
substrate or moveable stage.
[0051] FIGS. 7A and 7B are diagrams of components of a controller
module and its connections to certain components of the device,
including the adjustable tracking mirror(s) and substrate or
moveable stage position sensing devices. Connections are indicated
by arrows. A solid arrow indicates a signal sent from the
controller module to the respective component. Dotted arrow
indicates the path for measurement of velocity fluctuations of the
stage or substrate and translation into a driving signal that
controls the motor operably connected to an acceleration or motion
tracking mirror. Dashed arrows indicate the movement of light from
along an optical path among components of the optical scanning
system. FIG. 7A is related to a dual mirror embodiment, while FIG.
7B is related to a single mirror embodiment.
[0052] FIG. 8A provides a flowchart of a method of operating a dual
tracking mirror embodiment of a device to capture a stabilized
image of a field of a moving substrate.
[0053] FIG. 8B provides a flowchart of a method of operating a
single tracking mirror embodiment of a device to capture a
stabilized image of a field of a moving substrate.
[0054] FIGS. 9A and 9B illustrate an example of a pixel smear of an
image of +1 and +2 respectively. Each square represents a
pixel.
[0055] FIG. 10 illustrates an embodiment of the optical scanning
system, including components for controlling and storing data from
the system.
DETAILED DESCRIPTION
[0056] The details of various embodiments of the invention are set
forth in the description below. Other features, objects, and
advantages of the invention will be apparent from the description
and the drawings, and from the claims.
[0057] As used herein, the term "objective lens" refers to an
element or group of elements, in an optical scanning system, that
comprises one or more lenses and is configured and operative to
magnify an electromagnetic (e.g., such as optical) signal. In some
embodiments, an objective lens has a large numerical aperture (NA),
such as an NA in a range between 0.6 and 1.5 and performs imaging
via air immersion or liquid immersion (e.g., such as water, oil, or
other immersion fluids). In various embodiments, an objective lens
may have a focal length in the range from 2 mm to 25 mm.
[0058] As used herein, the term "substrate" refers to an object
having a multitude of distinct features that are targets for
imaging. These features may or may not be arranged in a spatially
uniform pattern. For example, in some embodiments a substrate
comprises a non-planar structure with a surface, such as a bead or
a well, to which target biomolecules have been attached as the
target features. In another example, in some embodiments a
substrate comprises an array chip. An array chip (i.e., an array,
microarray, or chip) refers to a solid phase support having a
surface, preferably but not exclusively a planar or substantially
planar surface, that carries attachment sites to which target
biomolecules (e.g., such as proteins or nucleic acids) have been
attached as the target features.
[0059] As used herein, the term "field" refers to an area of a
substrate capable of being captured within a single image by a
camera. A field on the substrate is related to the field of view of
the camera. An entire substrate may be scanned by taking images of
a plurality of fields on a substrate.
[0060] As used herein, the term "optical path" or "light path"
refers to the path of light or other electromagnetic radiation from
a source to the camera sensor. Manipulation of the optical path by
mirrors along the optical path enable the capture of a still image
from a continuously moving substrate with random or systematic
velocity fluctuations.
[0061] As used herein, the term "scanning" refers to operations to
observe and record the status of a substrate.
[0062] As used herein, the term "velocity tracking mirror" refers
to a mirror configured to track the movement of a substrate at a
velocity. This velocity may be fixed or variable. The velocity may
be predetermined, or may include systematic or random error in the
velocity.
[0063] As used herein, the term "velocity tracking error" refers to
an error in the tracking of a substrate or stage velocity by the
velocity tracking mirror. In some embodiments, this is the result
of a deviation in the velocity of the substrate from the velocity
being tracked by the velocity tracking mirror.
[0064] As used herein, the term "acceleration tracking mirror"
refers to a mirror that is operably connected to an optical
scanning system to rotate in response to a nonlinearity, such as a
systematic or random error in stage velocity, or any other
deviations from an expected or constant stage velocity. In some
embodiments, the acceleration tracking mirror is paired with a
velocity tracking mirror to provide a still image of a moving
substrate with reduced pixel smear.
[0065] As used herein, the term "electrical motor" refers to a
device that converts an electrical signal to a physical movement,
such as a motor that rotates in response to electrical energy. In
some embodiments, the electrical motor provides a rotation
mechanism for rotating a velocity tracking mirror or an
acceleration tracking mirror. The electrical motor can be operably
linked to a controller module that sends an electrical signal or
driving signal to effect controlled movement of the electrical
motor. An electrical motor may be a galvanometer or a piezoelectric
actuator. As used herein, a "galvanometer" refers to a coil in a
magnetic field that moves in response to an electrical signal. This
can act as an electrical motor to actuate rotary motion of a
tracking mirror. As used herein, the term "piezoelectric actuator"
refers to a type of electric motor based upon the change in shape
of a piezoelectric material when an electric field is applied.
Although electrical motors are referred to in this specification as
a preferred embodiment, other devices to provide actuation of
components of the parts of the invention described herein, such as
those based on hydraulics, pneumatics, or magnetic principles may
also be used.
[0066] As used herein, the term "controller module" refers to one
or more components in the device that provide control over
components of the optical scanning system. In particular, the
controller module includes devices that control movement of the
electrical motors operably connected to one or more tracking
mirrors. Thus, the controller module generates and transmits a
driving signal to these electrical motors. The driving signal may
be generated from a pre-programmed or observed stage or substrate
motion. The driving signal may be generated from information
collected by a position or velocity sensor, such as an encoder, and
used to generate a velocity measurement that is then translated
into a responsive driving signal to control movement of one or more
tracking mirrors.
[0067] As used herein, the term "electrical signal" or "driving
signal" refers to a controlled amount of energy sent to an
electrical motor that the motor transforms into physical movement.
For example, a galvanometer can affect rotation of a mirror to
track a moveable stage and to return to its original position after
imaging is complete by sending a drive signal that resembles a
sawtooth wave.
[0068] As used herein, the term "duty cycle" refers to the percent
of time a tracking mirror is tracking the stage and the camera is
imaging the field (as opposed to flyback time, where the tracking
mirror is returning to its initial position).
[0069] As used herein, the term "imaging frequency" or "image
capture frequency" refers to the frequency of image capture of
fields on a substrate.
[0070] As used herein, the term "pixel smear" refers to a measure
of the spread of a pixel along an axis due to movement of an imaged
object during image capture. A high amount of pixel smear will
generate an image that is less sharp and has a higher amount of
blur. In some embodiments, pixel smear is generated due to velocity
fluctuations that are not compensated for in the optical path or in
the movement of one or more tracking mirrors. Provided herein, in
some embodiments, are devices and methods for capturing an image of
a continuously moving substrate on a moveable stage with velocity
fluctuations wherein the amount of pixel smear along the primary
axis of movement of the substrate is mitigated by the rotation of
one or more tracking mirrors along the optical path.
[0071] As used herein, the term "logic" refers to a set of
instructions which, when executed by one or more processors (e.g.,
CPUs) of one or more computing devices, are operative to perform
one or more functionalities and/or to return data in the form of
one or more results or of input data that is used by other logic
elements and/or by elements that control the operation of
mechanical devices (e.g., such as servos and the like). In various
embodiments and implementations, any given logic may be implemented
as one or more software components that are executable by one or
more processors (e.g., CPUs), as one or more hardware components
such as Application-Specific Integrated Circuits (ASICs) and/or
Field-Programmable Gate Arrays (FPGAs), or as any combination of
one or more software components and one or more hardware
components. The software component(s) of any particular logic may
be implemented, without limitation, as a standalone software
application, as a client in a client-server system, as a server in
a client-server system, as one or more software modules, as one or
more libraries of functions, and as one or more static and/or
dynamically-linked libraries. During execution, the instructions of
any particular logic may be embodied as one or more computer
processes, threads, fibers, and any other suitable run-time
entities that can be instantiated in the hardware of one or more
computing devices and can be allocated computing resources that may
include, without limitation, memory, CPU time, storage space, and
network bandwidth.
Optical Scanning System and Methods of Use
[0072] Provided herein is a lightweight, cost-effective system for
high frame rate image capture of portions or fields of a substrate
with a high sensitivity while the substrate is moving on a moveable
stage. This optical scanning system is capable of high speed,
single molecule, single-fluor imaging, which to date has only been
provided by heavy and expensive systems requiring precise control
of stage movement, or through slower step and repeat optical
scanning systems. The optical scanning system provided herein can
be used as an image on the fly system (continuously moving stage)
by using scanning optics which compensate for stage velocities that
vary by 1% to 10% (typically resulting in image blur of at least
several pixels). This compensation can result in an image
equivalent of a tracked staged velocity with fluctuations less than
0.1% or a pixel smear of an image of no more than +/-1 pixel.
Therefore, the scanning optics disclosed herein provide a system to
compensate for velocity error (i.e., velocity fluctuations), such
as localized accelerations and decelerations of a moveable stage or
substrate, to provide a stabilized image field to a camera during
imaging of a continuously moving moveable stage to reduce pixel
smear.
[0073] The optical scanning system disclosed herein uses rotatable
scanning optics and a control system to stabilize an optical path
between a substrate and a detector while a substrate is in motion.
The rotate able scanning optics rotate in response to stage
velocity and stage velocity fluctuations (or to substrate velocity
and substrate velocity fluctuations). Scanning optics provided by
one embodiment of an optical scanning system with dual tracking
mirrors are shown in FIG. 1. In this embodiment, the optical
scanning system comprises a moveable stage 110 configured to move a
mounted substrate 120 along an axis. The substrate 120 comprises
one or more fields 121 that are individually imaged by the optical
scanning system as the stage is continuously moving. The substrate
is illuminated by an illumination mechanism (not shown), and light
from the substrate travels along an optical path through the
objective lens 130. The image of the moving substrate is stabilized
with respect to an image sensor by a velocity tracking mirror 140
and an acceleration tracking mirror 150. An image of the field 121
is captured by a camera 160 comprising an image sensor. The
velocity tracking mirror 140 is configured to rotate about an axis
parallel to the plane of the image field. The rotation of the
velocity tracking mirror 140 adjusts the optical path to stabilize
the image of a field moving at a predetermined velocity during an
image capture by the camera 160. The acceleration tracking mirror
150 is configured to rotate about an axis parallel to the plane of
the image field. The acceleration tracking mirror 150 rotates as a
function of velocity fluctuations (i.e., accelerations) of the
moving stage or substrate. This rotation adjusts the optical path
to stabilize an image by compensating for velocity fluctuations in
the movement of the stage and/or substrate along an axis.
[0074] The optical scanning system in several embodiments is
configured to image a continuously moving object, such as a
substrate mounted on a moveable stage, in a scanning fashion. In
such embodiments, a substrate is typically mounted (or otherwise
placed) on a moveable stage that is coupled to one or more
mechanisms (e.g., motors or other actuators) that can continuously
move the substrate under an objective lens while a camera captures
an image of a field of the substrate. The moveable stage is
configured and operative to move the substrate along a direction
that is normal to the optical axis of the objective lens. In some
embodiments, the axis of movement of the moveable stage is
orthogonal to the operation of autofocus-types of mechanisms, which
generally move an imaged object and/or an objective along the
optical axis of the objective lens.
[0075] In various embodiments, the velocity of the moveable stage
may be in a range from 0.1 mm per second to 1000 mm per second (or
greater). In some embodiments, the velocity of the moveable stage
may be in a range from 10 mm per second to 100 mm per second. In
some embodiments the moveable stage (and therefore the substrate
mounted thereon) can be configured to move at a constant velocity,
although the stage is still subject to velocity fluctuation errors
that are compensated for by the optical systems provided herein. In
some embodiments, the moveable stage moves at a velocity of 10 to
50 mm per second. In some embodiments, the velocity of the moveable
stage is about 25 mm per second. In other embodiments, the moveable
stage can be configured to move with non-constant velocity. This
non-constant velocity can also be subject to fluctuation errors
that are compensated for by the optical systems provided
herein.
[0076] In some embodiments, mechanisms may be used to facilitate
the motion of the moveable stage at a given desired velocity. Such
mechanisms may comprise one or more components that cause motion
(e.g., such as linear motors, lead screws, screw motors, speed
screws, etc.) and one or more components (e.g., such as various
types of bearings) that reduce friction.
[0077] For example, in some embodiments, a moveable stage may use
metal bearings (e.g., such as ball bearings, cylinder bearings,
cross-roller ball bearings, etc.) that have repeatability of
several microns to facilitate motion of the moveable stage at a
given desired velocity. Repeatability is fundamentally the effect
of rolling a metal bearing in oil--as the metal bearing rolls it
bounces, and such bouncing introduces jitter in the motion of the
object that is being moved on the bearings. The "repeatability" of
such motion can be uniform only above a certain range because any
two metal bearings can bounce in the same way only within a certain
tolerance. Thus, embodiments that use ball bearings typically have
greater velocity fluctuations, and thus introduce image blur (e.g.,
pixel smear). However, stages using ball bearings provide several
advantages, including that they are lighter, smaller, and cheaper
than comparable air bearing stages. Thus, provided herein according
to some embodiments are improved scanning optics to reduce image
blur or pixel smear due to moveable stage velocity fluctuations,
including stages with ball bearings or other components that
provide motion subject to some velocity fluctuations.
[0078] In some embodiments, the velocity of the moveable stage
fluctuates from the intended velocity by more than 0.1% during
continuous optical scanning. In some embodiments, the velocity of
the moveable stage fluctuates from the intended velocity by more
than 0.5% during continuous optical scanning. In some embodiments,
the velocity of the moveable stage fluctuates by between 0.1% and
1% during continuous optical scanning. In some embodiments, the
optical scanning system provided herein reduces an image blur or
pixel smear from a moveable stage with a velocity fluctuation of
between 0.1% and 1% to less than 0.1%. In some embodiments, the
pixel smear for a stabilized image is less than +/-1 pixel. In some
embodiments, the moveable stage is configured to move a substrate
in a continuous motion in a first known lateral direction with
respect to the objective lens while a camera with a two dimensional
full-frame electronic sensor produces the two-dimensional image. In
some embodiments, the moveable stage is configured to move in a
continuous serpentine fashion to image a plurality of rows or
columns of fields on a substrate.
[0079] In some embodiments, a substrate is mounted (or otherwise
placed) on a moveable stage. In some embodiments, the substrate
comprises an array having target biomolecules disposed thereon. In
some embodiments, the substrate comprises a multitude of distinct
features that are targets for imaging. e.g., such as array chips.
In some embodiments, the substrate comprises a randomly positioned
array of targets for imaging.
[0080] In some embodiments, the substrate comprises a multitude of
distinct features that are targets for imaging. For example, in
some embodiments a substrate comprises a non-planar structure with
a surface, such as a bead or a well, to which target biomolecules
have been attached as the target features. In some embodiments, a
substrate comprises an array chip. In some embodiments, the array
chip is a solid phase support having a surface, e.g., a planar or
substantially planar surface, that carries attachment sites to
which biomolecules are attached as the target features. In some
embodiments, the attachment sites on the array chip may be arranged
in an ordered pattern or in random fashion. In some embodiments,
the attachment sites are configured to have dimensions suitable for
the attachment of target biomolecules. An attachment site is thus
spatially defined and is not overlapping with other sites; that is,
the attachment sites are spatially discrete on the array chip. When
attached to the attachment sites, the biomolecules may be
covalently or non-covalently bound to the array chip.
[0081] In some embodiments, the substrate is a biochip. In some
embodiments, the biochip comprises high throughput microfluidics.
In some embodiments, the biochip comprises biomolecules for
detection of single molecules from a sample. In some embodiments,
the substrate comprises an array having target nucleic acids
disposed thereon. In another embodiment, the substrate comprises a
multitude of distinct features that are targets for imaging.
[0082] In some embodiments the attachment sites on a substrate are
divided into fields that are each imaged separately. A typical
substrate may be divided into hundreds or thousands of fields that
are arranged in a rectangular pattern of rows and columns. (For
example, the rows and columns of fields may include track regions
that are aligned substantially along a horizontal dimension and a
vertical dimension, respectively).
[0083] In such embodiments, the techniques described herein provide
for scanning and imaging a substrate field by field. In one
example, an optical scanning system images a substrate in a
scanning fashion (as described herein) while the moveable stage is
moving the substrate along a y-direction in a plane and/or axis
that is substantially normal to the optical axis of the objective
lens. In this example, the optical scanning system ceases imaging
when the end of the column of field(s) being imaged is reached in
order to allow the moveable stage to position the substrate for
imaging of the next column of field(s). In another example, an
optical scanning system images a substrate in a scanning fashion
(as described herein) while the moveable stage is moving the
substrate backward and forward in a serpentine fashion (e.g., along
a y-direction) in a plane that is substantially normal to the
optical axis of the objective lens. In this example, the optical
scanning system images a column of field(s) while the moveable
stage is moving the substrate in one direction and then images the
next/adjacent column of field(s) while the moveable stage is
moving/returning the substrate in the opposite direction, e.g., the
optical scanning system images the substrate by effectively
traversing the columns of fields in a continuous serpentine
fashion.
[0084] The objective lens of the optical scanning system is
configured and operative to image a substrate or a portion thereof
onto the camera. In some embodiments, the objective lens is an
element or group of elements, in an optical scanning system, that
comprises one or more lenses and is configured and operative to
magnify an electromagnetic (e.g., such as optical) signal. In some
embodiments, an objective lens has a large numerical aperture (NA)
(e.g., NA in a range between 0.6 and 1.5) and performs imaging via
air immersion or liquid immersion (e.g., such as water, oil, or
other immersion fluids). In various embodiments, an objective lens
may have a focal length in the range from 2 mm to 40 mm. The
objective lens can be an off-the-shelf microscope objective or a
custom-designed, multi-element optical component. In some
embodiments, the objective lens is configured to image at least a
two-dimensional portion of a substrate onto the two dimensional
full-frame electronic sensor of the camera to produce a
two-dimensional image.
[0085] The magnification of an objective lens is the ratio of the
size of an image space pixel (i.e., a camera pixel) to the actual
size of the object space area that corresponds to the image space
pixel as observed by the camera. For example, a magnification of
16.times. allows a camera using 81.tm pixels to observe 500 nm
object space pixels. In some embodiments, the objective lens has a
magnification from 4.times. to 100.times.. In some embodiments, the
objective lens has magnification of 20.times. to 50.times.. In some
embodiments, the objective lens has a magnification of
40.times..
[0086] In some embodiments, the objective lens is operably
connected to an electrical motor for positioning the objective lens
to allow auto-focusing. In some embodiments, the device comprises a
focusing sensor. In some embodiments, the device comprises an array
of focusing sensors.
[0087] In some embodiments, auto-focus mechanisms used are based on
optical sensing methods. In some embodiments, auto-focusing is
performed by image content analysis. In some embodiments,
autofocusing is performed by obtaining multiple images of the
substrate at multiple focal distances, determining an optimal focal
distance for each of the images, and using a feedback loop to
adjust the focal distance.
[0088] Autofocusing can be performed by directing a laser beam at
the substrate, measuring a reflection of the laser beam off the
substrate to provide a reference point, and using a feedback loop
to adjust the focal distance. In some embodiments, non-optical
types of non-contact position sensors are used. These sensors are
capable of making position readings with high bandwidth and a
tracking precision of 0.1 p.m. or less. In some embodiments,
capacitive position sensors may be used (see, e.g., US
2002/0001403, whose disclosure is incorporated herein by
reference).
[0089] In some embodiments, autofocus of the objective lens is
achieved in less than 100 ms. In some embodiments, the range of
autofocus provided by the device is +/-200 p.m.
[0090] In some embodiments, the optical scanning device comprises
an active autofocus system that measures distance to the subject
independently of the optical system, and subsequently adjusts the
objective lens to correct focus. In some embodiments, a passive
autofocus system that determines correct focus by performing
passive analysis of the image that is entering the optical system
is used. Passive autofocusing can be achieved, for example, by
phase detection or contrast measurement.
[0091] In some embodiments, the optical scanning system comprises a
camera capable of capturing a 2-dimensional still image of a field
of the substrate while the substrate is being moved by the moveable
stage. In some embodiments, the optical scanning system comprises a
full-frame camera. In some embodiments, the full-frame camera is a
Complementary Metal-Oxide Semiconductor (CMOS) camera. These full
frame cameras have high speed, high resolution, and low cost.
Furthermore, they are compatible with the optical scanning system
for capturing an image of a continuously moving substrate at a high
resolution. In some embodiments, the camera is a scientific CMOS
(sCMOS) camera. In some embodiments, the camera is a non-CMOS
camera capable of operating in full-frame mode.
[0092] The optical scanning systems described herein are configured
to use fast cameras in conjunction with a scanning optics (e.g.,
single mirror or dual mirror embodiments) in order to achieve
continuous exposure of a still image while the substrate being
imaged is moving. In some embodiments, the size (length and/or
width) of a camera pixel is in a range from 5 .mu.m to 10 p.m.,
preferably but not exclusively in the range of 6-8 p.m. In some
embodiments, the size of a camera pixel is 6.5 p.m. In some
embodiments, the camera comprises an imaging sensor on the range of
15.times.15 mm to 10.times.10 mm.
[0093] In various embodiments, the optical scanning systems
described herein are configured to scan a continuously moving
substrate (e.g., such as an array chip) by using fast cameras that
do not move the image through the camera, e.g., such as non-TDI
cameras and other cameras (including TDI cameras) that operate in
full-frame 2D mode. CMOS cameras are an example class of such
cameras. CMOS cameras typically use an active-pixel sensor (APS)
that is an image sensor comprising of an integrated circuit
containing an array of pixels, where each pixel includes a
photodetector and an active amplifier.
[0094] A high-speed camera may be defined in terms of the number of
pixels that the camera can expose in a unit of time. For example,
the speed of the camera may be defined by the mathematical product
of the number of pixels in the field of view and the frames per
second that the camera can take. Thus, a camera with a field of
view of 5.5 megapixels (e.g., a view of 2560 pixels by 2160 pixels)
running at 100 frames per second (fps) would be able to expose 550
megapixels per second; thus, such camera is termed herein as a
"550" megapixel camera. Examples of such cameras include, without
limitation, CMOS, sCMOS, and similar cameras. In various
embodiments, the optical scanning systems described herein may use
cameras in the range from 10 megapixels to 2500 megapixels. In some
embodiments, the camera comprises a 2-dimensional, full frame
electronic sensor.
[0095] Scanning optics described herein as part of the optical
scanning system can include single tracking mirror and dual
tracking mirror embodiments having one or more rotatable mirrors
affixed along an optical path of the system between the imaged
object and the camera. In a dual tracking mirror embodiment, two
sets of scanning optics are used, each able to move in concert to
track the motion of a moveable stage along an axis during imaging.
A first scanning optic (e.g., a velocity tracking mirror) is used
to track the movement of a stage at an anticipated velocity or
velocity pattern to enable imaging of a field by a camera while the
field is in motion. A second scanning optic (e.g., an acceleration
tracking mirror) is used to compensate for local stage
accelerations that could result in unacceptable pixel smear, thus
stabilizing the image. In single tracking mirror embodiments, a
single set of scanning optics is used both to track the movement of
a stage at an anticipated velocity or velocity pattern and to
compensate for local stage accelerations (i.e. velocity
fluctuations) that could result in unacceptable pixel smear, thus
stabilizing the image. For single tracking mirror embodiments, a
single set of scanning optics compensates for all stage motion
including velocity and acceleration (or velocity fluctuations). In
some embodiments, the single set of scanning optics includes a
motion tracking mirror to indicate its compensation for both
constant or anticipated velocity or velocity patterns and measured
or predetermined velocity fluctuations (accelerations).
[0096] In some embodiments, the movement of a tracking mirror in
response to velocity fluctuations of the moveable stage is based on
a feedback control mechanism. In some embodiments, the feedback
control mechanism comprises a device to measure position of a
substrate over time, such as an encoder. In some embodiments, the
movement of a mirror in response to velocity fluctuations is based
on predetermined velocity fluctuations for a moveable stage. In
some embodiments, all rotatable scanning optics are positioned
along an optical path before any splitter used to split an image to
multiple cameras.
[0097] In some embodiments, provided herein are optical scanning
devices comprising a velocity tracking mirror configured to rotate
to allow a camera sensor to image a field of a substrate moving
along an axis on a moveable stage. The velocity tracking mirror is
operably mounted to the device to reflect light along an optical
path from the objective lens to the camera.
[0098] In order to maintain a still image of a moving substrate,
the velocity tracking mirror is configured and operative to move in
coordination with the moveable stage, while the moveable stage
moves the substrate in the same specified direction, in order to
reflect light from the objective lens to the camera. Thus, the
velocity tracking mirror can be operably mounted to the device to
rotate about a fixed axis. In some embodiments, the fixed axis is
parallel to the plane of the 2-dimensional substrate image. In some
embodiments, the fixed axis is orthogonal to the optical path.
Thus, the velocity tracking mirror is configured and operative to
perform an angular motion that allows the camera to acquire a still
image of a field of the substrate through an objective lens while
the substrate is being moved by the moveable stage.
[0099] The velocity tracking mirror can be operably coupled to an
electrical motor to effect rotation of the velocity tracking
mirror. In preferred embodiments, the electrical motor operably
coupled to the velocity tracking mirror is a galvanometer, although
other types of electrical motors may be used. An example of a
suitable galvanometer is a Nutfield QS-7 OPD Galvanometer Scanner
(Nutfield Technology). In some embodiments, other mechanisms to
actuate the velocity tracking mirror, such as those based on
hydraulics, pneumatics, or magnetic principles, may also be used.
In some embodiments, the electrical motor is operatively coupled to
the velocity tracking mirror and is operative to angularly move the
velocity tracking mirror in coordination with the moveable stage,
while the moveable stage moves the substrate, in order to keep an
image of the substrate (or a field) still with respect to the
camera while the image is being acquired through the objective
lens.
[0100] The movement of the velocity tracking mirror can be
coordinated through a controller module configured to send a
driving signal to the electrical motor operably connected to the
velocity tracking mirror. The controller module can include a
motion controller component to generate a desired output or motion
profile and a drive or amplifier component to transform the control
signal from the motion controller into an electrical signal or a
drive signal that actuates the electrical motor.
[0101] In some embodiments, the velocity tracking mirror has an
angular range of rotation of about 60 degrees, 50 degrees, 40
degrees, 30 degrees, 20 degrees, 15 degrees, 10 degrees or 5
degrees. In some preferred embodiments, the velocity tracking
mirror has an angular range of rotation of about 3 degrees, 2
degrees, 1 degree, 1/2 degree, 1/4 degree, or 1/10 degree.
[0102] In an optical scanning system that uses a velocity tracking
mirror to image a moving substrate, the mirror angle is adjusted
with time so that a camera can view a fixed area on a moving
substrate. This is referred to as the "forward scan" time. The
velocity tracking mirror can then quickly rotate to return to its
initial position. This is referred to as a "fly-back" time or
"backscan" time. During the fly-back time, the image projected onto
the camera is not stable.
[0103] FIG. 2 illustrates a diagram of velocity tracking mirror
angular movement and timing according to an example embodiment. In
operation, the objective lens is focused on a substrate (e.g., an
array chip) that is moving along an axis during imaging. FIG. 2
shows movement of the stage over time 210. During this movement,
the velocity tracking mirror rotates from its initial position to
its end position to track the movement of the substrate, which is
represented as the forward scan time 220. During a single forward
scan, a portion of the substrate is imaged, which is referred to
herein as a field. The rotation of the velocity tracking mirror
allows imaging of the substrate portion corresponding to the field
by the camera during the exposure time, thereby allowing sufficient
exposure onto the camera sensor. Any remaining movement of the
field with respect to the camera can be due to velocity
fluctuations, or deviations of the substrate velocity from the
anticipated velocity. When the velocity tracking mirror reaches its
extreme end position, it then moves back to its initial position in
preparation for a new scan, which is represented by the waveform or
motion of the mirror at 230 (fly-back time). Still images of the
substrate are not acquired during the fly-back time intervals. The
forward scan and fly-back motions of the velocity tracking mirror
are represented as a sawtooth waveform (FIG. 2), which reflects
both the motion of the velocity tracking mirror during scanning and
flyback and the driving signal sent to an electrical motor operably
connected to the velocity tracking mirror to actuate the
mirror.
[0104] An embodiment of a sawtooth waveform to drive the mirror
(including a forward scan and backscan segments) is shown in FIGS.
2 and 3. In some embodiments, the velocity tracking mirror may have
a non-linear response over segments of it's range of motion. In
this case, the velocity tracking mirror response may be linearized
by adjusting the waveform or driving signal so that you to
linearize the response from the velocity tracking mirror.
[0105] In some embodiments, the velocity tracking mirror is
operably coupled to an electrical motor to effect rotation of the
acceleration tracking mirror. In preferred embodiments, the
electrical motor operably coupled to the velocity tracking mirror
is a galvanometer, or electric coil in a magnetic field that moves
in response to an electrical current. In some embodiments, other
mechanisms to provide actuation of the velocity tracking mirror,
such as those based on hydraulics, pneumatics, or magnetic
principles, may also be used. In some embodiments, the electrical
motor operatively coupled to the velocity tracking mirror is
operative to generate angular motion of the velocity tracking
mirror as a function of a velocity of the moveable stage or
substrate.
[0106] In some embodiments, the movement of the velocity tracking
mirror is coordinated through a controller module configured to
send a driving signal to the electrical motor operably coupled with
the velocity tracking mirror. The controller module can include a
motion controller component to generate a desired output or motion
profile and a drive or amplifier component to transform the control
signal from the motion controller into energy that is presented to
the electrical motor as an electrical signal or a drive signal.
[0107] In some embodiments, the driving signal or electrical signal
sent to the electrical motor operably coupled with the velocity
tracking mirror can be a linearized velocity tracking error
waveform defined as a function of G(O,w,c(0)), where G is a
modified triangle wave with O=angular position, w=frequency, and
6(0)=amplitude.
[0108] The movement of a tracking mirror can be characterized by
its duty cycle, defined as the portion of time the tracking mirror
is operably moving in the forward scan motion to allow active
imaging of the substrate. For example, if the tracking mirror
tracks the substrate to allow imaging by the camera during at least
90% of the tracking mirror cycle (e.g., when the tracking mirror
fly-back time is equal to or less than 10% of the cycle), then this
technique allows the camera to operate with at least a--90% overall
readout efficiency.
[0109] In some embodiments, such as fluorescence imaging where
longer exposure times may be needed, the scan time interval, during
which an image is collected by the camera, must be long enough to
build up adequate signal-to-noise ratios as fluorescence imaging
light levels are typically very weak.
[0110] The duty cycle is also impacted by the speed with which the
tracking mirror returns to its initial position. This fly-back time
interval can be configured to be only a small fraction of the
tracking mirror cycle, thus maximizing the duty cycle. For better
efficiency, the amount of time spent by a tracking mirror on each
imaged area is made commensurate with the camera's frame rate,
thereby allowing sufficient time to expose an image of each field
onto the camera.
[0111] In some embodiments, the duty cycle is greater than 60%. In
some embodiments, the duty cycle is from 60% to 90%. In some
embodiments, the duty cycle of the image capture is at least 1%,
2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%. In some embodiments, the
duty cycle can be as low as 10%, or can be in the range of 20%,
30%, 40%, 50%, 60%, 70%, 80% or 90%. In some embodiments, these
duty cycles are achieved with an imaging frequency of from 30 to
200 Hz. In some embodiments, these duty cycles are achieved with an
imaging frequency of from 30 to 40 Hz. In some embodiments, these
duty cycles are achieved with an imaging frequency of 30 Hz, 35 Hz,
40 Hz, 45 Hz, or 50 Hz.
[0112] During the fly-back time intervals, the optical scanning
system should cease imaging because the image being acquired is not
stable. Thus, in various embodiments various mechanisms can be used
to prevent image exposure to the camera during the fly-back time
intervals. For example, in some embodiments an acousto-optic
modulator (AOM) switch (or other type of fast switch) may be used
to turn on and off the illumination light that is incident onto the
substrate being imaged. In other embodiments, a suitable aperture
can be placed in the optical path of the illumination light, where
the illumination light is allowed to over scan, but the aperture
prevents the light from illuminating the substrate during the
fly-back time intervals by blocking out the light outside of the
field of view. In yet other embodiments, a suitable shutter can be
placed in the optical path of the illumination light, where the
shutter is kept open during exposure intervals and is closed during
the tracking mirror fly-back time intervals.
[0113] In dual tracking mirror embodiments, the optical scanning
device further comprises an acceleration tracking mirror configured
and operative to provide offset corrections to an optical path to
stabilize the transmission of light from a substrate to the camera
during imaging of the substrate (or a portion thereof). The offset
corrections are a function of velocity fluctuations in the movement
of the moveable stage along an axis as compared to the velocity
tracked by the velocity tracking mirror. These velocity
fluctuations can impact the accuracy of tracking of the moveable
stage by the velocity tracking mirror and result in an image with
unacceptable pixel smear. The rotation of the acceleration tracking
mirror, as provided herein, stabilizes the image of the field
captured by the camera to reduce pixel smear from velocity
fluctuations of the stage or substrate.
[0114] FIG. 3 provides one example of an acceleration tracking
mirror waveform generated in response to an average stage velocity
error for a field. When the stage velocity has a positive error, an
acceleration tracking mirror waveform is generated to track the
additional velocity of the stage. Conversely, when the stage
velocity has a negative velocity error, an acceleration tracking
waveform is generated to track the slower velocity of the stage
(i.e., it rotates in the opposite direction as a positive velocity
error). In some embodiments, the acceleration tracking mirror
waveform is generated and converted to a driving signal immediately
after sensing the velocity error. In some embodiments, the
acceleration tracking mirror waveform is generated based on an
average measurement of velocity during imaging of a field n-1, and
the driving signal is generated from this waveform to drive
movement of the acceleration tracking mirror during imaging of
field n.
[0115] Stage velocity error can be modeled as a function of
amplitude (A), stage position (x), and time (t), to give the
following function: [0116] F(A,x,t)=A(x)*Err(x,t)
[0117] In some embodiments, the electrical signal or driving signal
(D) to control movement of an electrical motor operably connected
to the acceleration tracking mirror can be determined based on the
stage velocity error by a function represented as follows: [0118]
D(F,C,x,E)=F(A,x,t)y*C*x+E,
[0119] where C is a scaling factor, x=stage position and E is an
offset. F(A,x,t)y is the average value of F(A,x,t) over the ramp
range=y, or over a prior field, as described herein. A function to
smooth discontinuities can also be used to generate the
acceleration tracking mirror driving signal.
[0120] In some embodiments, the acceleration tracking mirror is
operably coupled to an electrical motor to effect rotation of the
acceleration tracking mirror. In preferred embodiments, the
electrical motor operably coupled to the acceleration tracking
mirror is a piezoelectric actuator, although other types of
electrical motors may be used. In some embodiments, other
mechanisms to provide actuation of the acceleration tracking
mirror, such as those based on hydraulics, pneumatics, or magnetic
principles, may also be used. In some embodiments, the electrical
motor operatively coupled to the acceleration tracking mirror is
operative to generate angular motion of the acceleration tracking
mirror as a function of fluctuations in the velocity of the
moveable stage to compensate for velocity fluctuations during
imaging.
[0121] In some embodiments, the movement of the acceleration
tracking mirror is coordinated through a controller module
configured to send a driving signal to an electrical motor operably
connected to the acceleration tracking mirror. The controller
module can include a motion controller component to generate a
desired output or motion profile and a drive or amplifier component
to transform the control signal from the motion controller into
energy that is presented to the electrical motor as an electrical
signal or a drive signal. Since the movement of the acceleration
tracking mirror is a function of fluctuations in velocity of the
moveable stage, the controller module can further comprise a
position, velocity or acceleration sensor. This sensor can act as a
type of feedback sensor that determines information about the
position and/or motion of the substrate or moveable stage. In some
embodiments, the sensor comprises an encoder (e.g., a linear
encoder) or an interferometer operably mounted to the scanning
device. In some embodiments, the encoder is a non-interferometric
encoder. In some embodiments, an accelerometer could be used to
determine changes in velocity. In some embodiments, the sensor is a
component that provides information from a velocity fluctuation
table that includes anticipated velocity fluctuation values for a
stage to incorporate into the driving signal for the electrical
motor operably coupled to the acceleration tracking mirror.
[0122] An encoder can be a sensor, transducer or readhead paired
with a scale that encodes position. In some embodiments, the sensor
reads the scale (e.g., encoder counts) in order to convert the
encoded position into an analog or digital signal, which can then
be decoded into position by a digital readout (DRO) or motion
controller. Thus, in some embodiments, the position sensor
(including position, velocity, and/or acceleration sensors) is a
linear encoder that interfaces with encoder counts (or another
scale) on the substrate or moveable stage. In some embodiments, the
encoder counts on the substrate are positioned at a distance of 10
p.m., 5 .mu.m, 2 p.m., 1 .mu.m, or 500 nm or less between each
encoder count. In some embodiments, the resolution of position
detectable by the encoder is 1 nm or less. This can be done for
example, using interpolation between lines on a substrate or
between encoder counts. The spacing between encoder counts can
correlate with stage scan speed and frequency of position
measurement.
[0123] In some embodiments, the scale used by an encoder, such as a
linear encoder, can be optical, magnetic, capacitive, inductive,
based on eddy current. In some embodiments, position detection can
be done without a scale on the substrate or moveable stage, for
example, by using an optical image sensor based on an image
correlation method.
[0124] The position measurements from a substrate or stage position
or motion sensor are used to provide a set of data that represents
the measured velocity of the substrate or moveable stage. The
measured velocity can be compared with an anticipated velocity to
determine velocity fluctuations in the stage. These velocity
fluctuations can then be translated into an electrical signal
(e.g., a driving signal) which effects controlled movement of an
electrical motor operably connected to the acceleration tracking
mirror. The controlled movement of the acceleration tracking mirror
adjusts the position of the optical path between the substrate and
the camera to provide an image with increased stability, increased
sharpness, and/or reduced blur or pixel smear.
[0125] An electrical motor can be selected on the basis of its
ability to quickly respond to a driving signal comprising a
correction term based on measured velocity fluctuations. To provide
a quick response, in some embodiments, the electrical motor has a
total angular range of rotation of less than one degree. In some
embodiments, the electrical motor is a piezoelectric actuator or
another motor with a similar response time to the correction
signal. In some embodiments, the position sensor acquires position
information at a rate of equal to or greater than 500 Hz, 1 kHz, 2
kHz, 3 kHz, 4 kHz, 5 kHz, 10 kHz, 20 kHz, 50 kHz, 100 kHz and 250
kHz. In certain embodiments, a higher frequency of position
detection, e.g., 5 kHz or more, allows a more precise measurement
of the stage to increase the resolution of velocity fluctuation and
therefore provide a sharper image. However, lower frequencies may
be used that are sufficient to provide correction to prevent a
pixel smear of greater than two pixels.
[0126] For example, in some embodiments an encoder provides
substrate or stage position or motion measurement information to
logic executing in a computing device, such as a motion controller,
where the logic uses the measurement information to compute the
necessary correction term for the direction of stage movement and
to cause a servo mechanism, such as an electrical motor, to rotate
the acceleration tracking mirror based on the computed correction
term that is a function of velocity fluctuation of the moveable
stage.
[0127] The determination of velocity fluctuation can be determined
from two or more position measurements from the position sensor. In
some embodiments, near instantaneous velocity can determined from
the most recent 2 or 3 positions measured from the substrate.
[0128] In some embodiments, velocity fluctuation used to generate a
driving signal is determined from a pre-calculated table. A
velocity fluctuation may already be known for a stage, and can be
recorded into a table which is accessed by the motion controller
component. Thus, in these embodiments, the position sensor is a
component of the controller module that provides data from a
velocity fluctuation table to a motion controller.
[0129] By using an acceleration tracking mirror as described
herein, an optical scanning system can use a camera that operates
in a full-frame mode (e.g., such as a CMOS camera that does not
operate in TDI mode) to acquire still images of a moving substrate
within an accuracy of +/-one pixel. In some embodiments that are
employed for biological imaging, e.g., DNA sequencing or other
single molecule detection techniques, the extreme alignment
accuracy requirements of fluorescence imaging may necessitate the
use of at least one velocity and acceleration tracking mirror pair
to correct for movement, including velocity fluctuations, of a
substrate along an axis to remove nonlinearities in the motion of
the moveable stage.
[0130] In some embodiments, tracking of the movement of the stage,
including both a velocity of a stage and velocity fluctuations of
the stage along an axis, is performed by a single tracking mirror
(the single-mirror embodiment), referred to herein as a motion
tracking mirror. In this embodiment, a single motion tracking
mirror performs the functions of both the velocity and acceleration
tracking mirrors described above. Therefore, in a single mirror
embodiment, a drive signal is sent to an electrical motor operably
coupled to the single motion tracking mirror that is a function of
a predetermined stage velocity including both scanning and flyback
waveforms (e.g., a sawtooth wave) and is also a function of
velocity fluctuations of the stage or substrate, which can be
predetermined or can be based on one or more measurements that
provide information about the motion of the substrate or moveable
stage to determine a velocity fluctuation of the stage or
substrate.
[0131] Scanning optics provided in a single mirror embodiment of
the optical scanning system is shown in FIG. 4. In this embodiment,
the optical scanning system comprises a moveable stage 110
configured to move a mounted substrate 120 along an axis. The
substrate 120 comprises one or more fields 121 that are
individually imaged by the optical scanning system as the stage is
continuously moving. The substrate is illuminated by an
illumination mechanism (not shown), and light from the substrate
travels along an optical path through the objective lens 130. The
image of the moving substrate is stabilized with respect to an
image sensor by a motion tracking mirror 145. An image of the field
121 is captured by a camera 160 comprising an image sensor. The
motion tracking mirror 145 is configured to rotate about an axis
parallel to the plane of the image field. The rotation of the
motion tracking mirror 145 adjusts the optical path to stabilize
the image of a field during an image capture by the camera 160.
Rotation of the motion tracking mirror 145 is a function of both a
predetermined stage velocity and velocity fluctuations of the stage
or substrate. Thus, the single-mirror embodiment of the optical
scanning system provides a stabilized image with an improved
sharpness or reduced pixel smear over a system that does not
correct for stage velocity fluctuations while imaging a moving
substrate.
[0132] A controller module configured to drive the movement of the
single motion tracking mirror includes components of both a
controller module operably connected to a velocity tracking mirror
and components of a controller module operably connected to an
acceleration tracking mirror, as described in the two-mirror
embodiment above. Therefore, in some embodiments, the controller
module comprises a motion controller component that generates a
desired output or motion profile, a drive or amplifier component to
transform the control signal from the motion controller into an
electrical signal or drive signal. The controller module can also
include a position, velocity, or acceleration sensor configured to
determine the position or motion of the substrate or moveable
stage, and to send this signal to the motion controller component
to be used to generate the desired output or motion profile as a
function of the information from the sensor. The motion controller
component can then generate a motion profile for the single motion
tracking mirror that is a function of both the constant or
otherwise anticipated velocity of the substrate or stage (e.g., a
sawtooth waveform) and velocity fluctuations determined from the
signal from the sensor or that are predetermined for the stage.
Thus, the sawtooth waveform used to track velocity can be modified
according to a real time velocity measurement determined from a
signal from a positional sensor or from predetermined velocity
fluctuations.
[0133] FIG. 5 provides an example stage velocity error waveform
generated from data provided by a position, velocity or
acceleration sensor. Also shown are approximate velocity
corrections needed, otherwise known as the average velocity error
for a field. The next waveform (solid line) shows a velocity
tracking waveform modified by a correction term that is a function
of an average velocity error (e.g., an average velocity error for a
field). The dashed line represents a linearized, un-corrected
motion tracking mirror waveform (similar to the waveform used to
drive a velocity tracking mirror in the dual-mirror embodiment).
When there is a positive velocity error, the slope of the waveform
during scanning is increased, thereby increasing the speed of the
rotation of the mirror to compensate for the velocity error. When
there is a negative velocity error, the slope of the waveform is
decreased, thereby decreasing the speed of the rotation of the
mirror to compensate for the velocity error.
[0134] In some embodiments, the sensor determines an average
velocity of the stage or substrate based on a plurality of
measurements taken during imaging of a field. In some embodiments,
the sensor is a position sensor. In some embodiments, the position
sensor is an encoder (e.g., a linear encoder) or an interferometer
operably mounted to the scanning device. The signal from the
position sensor can be used to determine the average velocity of
the moveable stage or substrate using two or more of the most
recent positional measurements captured by the position sensor. In
some embodiments, these measurements can be used to adjust the
angle of the motion tracking mirror after sensing.
[0135] In some embodiments, the average velocity of a substrate or
stage is determined over a field n-1 and is used to provide a
correction term which is used for generating the motion profile of
the motion tracking mirror for field n. This is known as the field
level feed forward mechanism, as illustrated in FIG. 6. In some
embodiments of the field level feed forward correction mechanism,
the motion controller component generates a motion profile for
movement of the motion tracking mirror (in the single mirror
embodiment) or the acceleration tracking mirror (in the dual mirror
embodiment) as a function of the average velocity of the previous
imaged field. A field level feed-forward velocity tracking and
correction mechanism is distinct from other types of correction,
such as a scanline level feed forward mechanisms. Field-level feed
forward corrections are advantageous in that they reduce the
stringency of immediate signal processing while still providing
sufficient correction information to generate an acceptably sharp
image for monitoring information in single pixels (i.e., a pixel
smear of no more than +/-one pixel). Some image blur or pixel smear
may not be corrected by field level feed forward mechanisms,
however, in some embodiments, such as in single molecule imaging
applications (e.g., for biomolecular sensing), a pixel smear of up
to +/-one pixel is acceptable, and field-level feed forward
corrections can generate an acceptable smear when there is a
velocity fluctuation from field n-1 to field n that is within an
acceptable level (e.g., results in a pixel smear of no more than
+/-one pixel).
[0136] Provided in FIG. 6 is a diagram of an embodiment of field
level feed-forward correction. In this embodiment, velocity
tracking measurements of a chip or stage are obtained to generate a
mirror rotation drive signal that incorporates velocity
fluctuations of the moving stage. Here, the stage begins moving,
and positional information is obtained over time (or velocity
information is obtained) to determine a pre-field non-linearity of
the velocity of the stage (velocity fluctuations). When the first
field is imaged, a driving signal that is a function of the average
velocity measured in the pre-field stage is sent to the motion
tracking mirror (or acceleration tracking mirror in two-mirror
embodiments). For the next consecutive fields, the process is
repeated using velocity error determined from positional
information of the prior field (N-1) over time. The driving signal
is determined as a function of this velocity error and sent to the
motion tracking mirror for rotation during field N. FIG. 6 shows
stage velocity error over time and also the approximate velocity
error per field. The first arrow down from the stage velocity error
indicates that determination of an average velocity error from the
field, and the translation into a "same field" acceleration
correction waveform. The feed forward mechanism is indicated by the
second arrow down, translating this waveform to drive the mirror
for the next field n based on the waveform derived from field n-1.
In this manner, the stage velocity error is approximated for each
field based on the prior field n-1.
[0137] In one embodiment, the field level feed forward mechanism
proceeds according to the following steps: [0138] a) Measure
multiple positions of the substrate over field n-1. [0139] b)
Determine an average velocity for field n-1. [0140] c) Calculate
the velocity fluctuation for field n-1 and a correction term based
on this velocity fluctuation. [0141] d) Apply correction term to
motion profile (e.g., an electric motor waveform) to send to driver
or amplifier. [0142] e) Send a driving signal to an electric motor
operably linked to a motion tracking mirror or acceleration
tracking mirror to generate movement of the tracking mirror during
image capture of field n. [0143] f) Repeat process for remaining
fields in lane
[0144] In some embodiments, the total feedback loop in the
servomechanism based on field level feed-forward velocity tracking
is less than 100 ms, less than 90 ms, less than 80 ms, less than 70
ms, less than 60 ms, less than 50 ms, less than 40 ms, less than 30
ms, less than 20 ms, less than 10 ms, less than 5 ms, or less than
2 ms. In some embodiments, feed-forward velocity tracking is used
to adjust the movement of an acceleration tracking mirror in the
two mirror optical path alignment correction embodiment.
[0145] In some embodiments, in order to minimize error due to the
linear ramp of an electric motor-controlled single mirror, the
electric motor driving signal or waveform is adjusted to compensate
for systematic errors in tracking. Minimization of error in
generating a linear ramp (e.g., a forward scan or fly-back) of an
electric motor can also be achieved by reducing the speed of motion
of the mirror, such as by reducing the imaging frequency of the
optical scanning system. In some embodiments, the frequency of the
sawtooth waveform to control the electric motor in the single
mirror embodiment is kept at or below 200 Hz. In some embodiments,
the frequency of the sawtooth waveform to control the electric
motor in the single mirror embodiment is from 50 Hz to 30 Hz. In
some embodiments, the frequency of the sawtooth waveform to control
the electric motor in the single mirror embodiment is from 45 Hz to
35 Hz. In some embodiments, the duty cycle of the sawtooth waveform
to control the electric motor in the single mirror embodiment is
70% or less. In some embodiments, the duty cycle of the sawtooth
waveform to control the electric motor in the single mirror
embodiment is from 60% to 80%. In some embodiments, the frequency
of image capture and the duty cycle in the single mirror embodiment
are adjusted to have a total velocity tracking error of less than
2%. In some embodiments, the frequency of image capture and the
duty cycle in the single mirror embodiment are adjusted to have a
total pixel smear of less than 2 pixels or less than 1 pixel.
[0146] As discussed herein, according to some embodiments, the
controller module refers to a collection of components including i)
sensors to determine states of parts of the optical scanning system
(e.g., a stage position sensor) for feedback control, ii)
mechanisms that calculate or otherwise provides waveforms for
effecting movement of components of the optical scanning device
(e.g., a sawtooth wave to drive a velocity tracking mirror), or
iii) mechanisms that send a driving signal to an actuator based on
the waveform to effect movement of a component.
[0147] For example, as discussed above, the controller module can
be used to create the correct waveforms to drive the movement of
certain components, such as rotatable mirrors to adjust the optical
path, and synchronize them to stage motion based on stage encoder
or master clock values. The waveform for a velocity tracking mirror
can be a sawtooth waveform with a ramp that tilts the velocity
tracking mirror at the right speed to match of the velocity of the
stage. The waveform sent to an acceleration tracking mirror or to a
single rotatable mirror in the single mirror embodiment must
include a term to correct for velocity fluctuations that occur in
the moveable stage velocity. This waveform can be created by
"mapping" out the stage velocity non-linearities using a reticle
with calibration marks on it, or it can be created by taking the
measured stage velocity from the previous field, creating a
waveform that compensates for velocity non-linearities and using
that waveform to correct for velocity fluctuations in the next
field, i.e., the field level "feed-forward" approach. The waveform
can also be created by providing information from a velocity
fluctuation table to the controller module.
[0148] According to the techniques described herein, one or more
computing devices and/or various logic thereof are configured and
operative to control the coordinated motions of the scanning mirror
or mirrors (e.g., the acceleration and velocity tracking mirrors)
and the moveable stage. Thus, in some embodiments the moveable
stage (and therefore the substrate mounted thereon) can be
configured to move with constant velocity, in which case the
back-scan motion of the tracking mirror will also be at a suitable
constant velocity. In other embodiments, the moveable stage can be
configured to move with non-constant velocity, in which case the
back-scan motion of the tracking mirror will also be at a suitable
non-constant constant velocity.
[0149] The controller module can also be used to synchronize
components of the optical scanning device to enable capture of an
image of a field of a substrate on a moving stage. In addition to
linking motion of the rotatable mirrors to the velocity of a
moveable stage, the controller module can also control other
components of the device. In some embodiments, the controller
module comprises a mechanism to control illumination of the field.
For example, the controller module may send a signal to an
illumination device, such as a laser, to time illumination with the
image capture process. In some embodiments, illumination state is
dependent upon the sawtooth waveform sent to a velocity tracking
mirror. In some embodiments, the controller module sends a signal
to control movement of the moveable stage at a selected velocity or
along a selected path, such as a serpentine path to image several
fields on a substrate.
[0150] The connection of the controller module to certain
components of an optical scanning system, according to a
dual-mirror embodiment, is shown in FIG. 7A. As illustrated in this
embodiment, the controller module is operably connected to an
illumination component to control illumination of a substrate, such
as by timing illumination with image capture timing. The controller
module is operably connected to a camera to control image capture
by the camera to coordinate with the motion of the rotating
mirrors, e.g., such that an image is acquired during tracking of
each field, and no image is acquired during the fly-back period of
a tracking mirror. As described in more detail herein, the
controller module can comprise a memory, a processor, and a driver.
The memory can hold a predetermined velocity or velocity
fluctuation information to be used by the processor to generate a
waveform. The memory can also hold a predetermined waveform. The
waveform can be sent to the driver to generate a driving signal. In
some embodiments, the controller module is operably connected to an
encoder (e.g., a linear encoder) to receive positional information
about the moving stage over time. The controller module can then
generate a drive signal as a function of velocity fluctuation from
the information from the linear encoder, which can then be sent to
the driver to send a driving signal to an acceleration tracking
mirror (or substrate tracking mirror in the one-mirror embodiment
(FIG. 7B)). The path from data collection from the stage to
movement of a tracking mirror is indicated by the dotted arrows,
which also include a driving signal sent from the controller module
to a motor operably connected to an acceleration tracking mirror
150 or motion tracking mirror 145. FIGS. 7A and 7B also depicts the
optical path of light from an illumination source to detection by
the camera according to a dual-mirror embodiment. Solid lines (not
arrows) in FIGS. 7A and 7B indicate an operable connection between
a motor and a component of the device actuated by the motor.
[0151] In an example embodiment, the optical scanning system
further comprises an illumination light source. In various
embodiments, the illumination source can emit light of various
wavelengths that are compatible with various fluorophores that can
be used in biomolecular detection, for example, light of wavelength
in a range from 400 nm to 800 nm. In some embodiments, the
illumination source is mounted underneath the substrate, such that
light collected by the objective lens is transmitted through the
field to the objective lens. In other embodiments, the illumination
source is mounted above the substrate, such that light collected by
the objective lens is reflected by the field to the objective
lens.
[0152] The optical scanning system can further comprise a dichroic
mirror. In an example embodiment, the optical scanning system
further comprises an illumination source and a dichroic mirror,
where the dichroic mirror is configured and operative at least to:
(a) reflect light from the illumination source to illuminate a
field of the substrate or a portion thereof; and (b) pass through
light that is emitted by the sample and passes through the
objective lens.
[0153] In some embodiments, the optical scanning system further
comprises a splitter. The splitter can be placed along the optical
path after the acceleration and velocity tracking mirrors (or
single tracking mirror) to split the optical signal comprising the
field image to two or more cameras.
[0154] The optical scanning system can also comprise a tube lens
component positioned in an optical path between the tracking mirror
and the objective lens, so that the tracking mirror can be situated
at the pupil of the objective lens. Relay lenses or tube lenses may
also be used along the optical path at other locations to invert an
image or to extend the optical path.
[0155] In some embodiments, the optical scanning system comprises a
relay lens system used to create a region in the optical path which
has all rays nominally parallel and also has a small beam diameter.
In some embodiments, scanning optical elements are placed where the
optical path has a small beam diameter to ensure that their
placement: (i) minimizes power loss, (ii) minimizes image
degradation and (iii) minimizes the size of the optical elements so
that their mass can be as small as possible. This enables higher
scanning frequencies and a lighter weight system.
[0156] The use of a relay lens system can facilitate
fluorescence-based optical scanning systems that are used for
biomolecular detection on a substrate, as these systems typically
employ very low light levels with dim fluorescence images. Thus,
relay lenses are effective to increase the efficiency and
sensitivity of the optical scanning system to keep image
acquisition time to a minimum. Further, in some embodiments,
illumination intensity must remain below the point where it can
damage biomolecules on the substrate.
[0157] FIG. 8A illustrates an example method for imaging a
substrate according to a dual-mirror embodiment. The method in FIG.
8A is not limited to being performed by any particular type of
machine or device, and therefore the method description hereinafter
is to be regarded in an illustrative rather than a restrictive
sense.
[0158] In step 810, a moveable stage moves a substrate under an
objective lens in a plane that is normal to the optical axis of the
objective lens. While the substrate is in motion, in step 820, a
servo mechanism (e.g., an electric motor) changes the angle of a
velocity tracking mirror to track the velocity of the moving stage
during the capture of an image of a field of the substrate. In some
aspects, a controller module that is part of or coupled to, the
velocity tracking mirror executes logic that controls the servo
mechanism operably connected to the velocity tracking mirror. In
step 840 a servo mechanism changes the angle of an acceleration
tracking mirror to track velocity fluctuations of the moving stage
during the capture of an image of a field of the substrate. In some
aspects, a controller module that is part of or coupled to, the
acceleration tracking mirror executes logic that controls the servo
mechanism in coordination with the moveable stage. In some
embodiments, logic receives feedback control information that
represents the movement (e.g., velocity fluctuations) of the
moveable stage and uses this information to adjust the input signal
to the servo mechanism, which in turn changes the angle of the
acceleration tracking mirror, thereby synchronizing the combined
motion of the velocity tracking mirror and acceleration tracking
mirror with the movement of the moveable stage. In some aspects,
this feedback information is received 831 from an linear controller
that detects whether there are any nonlinearities in the motion of
the moveable stage 830. The logic then uses this information to
compute offset corrections and passes the offset corrections as an
input signal to a servo mechanism that controls the angle of the
acceleration tracking mirror in the optical path between the
tracking mirror and the camera. In this manner, by making minor
adjustments to the angle of the acceleration tracking mirror, the
logic effectively removes from the image being acquired any errors
that are caused by nonlinearities in the motion of the moveable
stage.
[0159] In step 850, the camera records the still image of the
substrate (or a portion thereof) while the substrate is being moved
by the moveable stage.
[0160] FIG. 8B illustrates an example method for imaging a
substrate according to a single-mirror embodiment. The method in
FIG. 8B is not limited to being performed by any particular type of
machine or device, and therefore the method description hereinafter
is to be regarded in an illustrative rather than a restrictive
sense.
[0161] In step 810, a moveable stage moves a substrate under an
objective lens in a plane that is normal to the optical axis of the
objective lens, where the substrate comprises a multitude of
distinct features that are the targets of the imaging.
[0162] While the substrate is in motion, in step 845 a servo
mechanism changes the angle of a motion tracking mirror to track
velocity fluctuations of the moving stage during the capture of an
image of a field of the substrate. In some aspects, a controller
module that is part of or coupled to, the motion tracking mirror
executes logic that controls the servo mechanism in coordination
with the moveable stage. In some embodiments, logic receives
feedback control information that represents the movement (e.g.,
velocity fluctuations) of the moveable stage and uses this
information to adjust the input signal to the servo mechanism,
which in turn changes the angle of the motion tracking mirror to
compensate for velocity fluctuations of the moveable stage. In some
embodiments, the controller module incorporates the velocity
fluctuation of the moveable stage into a sawtooth waveform for
tracking a predetermined velocity, which is used as a driving
signal to control movement of the motion tracking mirror. In some
aspect, this feedback information is received 831 from an linear
controller that detects whether there are any nonlinearities in the
motion of the moveable stage 830. The logic then uses this
information to compute offset corrections and passes the offset
corrections as an input signal to a servo mechanism that controls
the angle of the motion tracking mirror in the optical path. In
this manner, by making minor adjustments to the angle of the motion
tracking mirror, the logic effectively removes from the image being
acquired any errors that are caused by nonlinearities in the motion
of the moveable stage.
[0163] In step 850, the camera records the still image of the
substrate (or a portion thereof) while the substrate is being moved
by the moveable stage.
[0164] Optical scanning systems provided herein compensate for
stage velocity (or any other imaging of moving components)
non-linearities (e.g., local stage accelerations) that would
normally result in a blurry image in a device that tracks only
stage velocity, but does not have a mechanism to compensate for
stage velocity fluctuations. In some embodiments, the optical
scanning system is capable of generating stabilized images of a
continuously moving substrate or other object at 30 frames per
second. In some embodiments, the optical scanning system is capable
of generating still images of a continuously moving substrate or
other object at from 10 to 30 frames per second. In some
embodiments, the optical scanning system is capable of generating
still images of a continuously moving substrate or other object at
40 frames per second. In some embodiments, the optical scanning
system is capable of generating still images of a continuously
moving substrate or other object at more than 30 frames per second,
40 frames per second, 50 frames per second, 60 frames per second,
70 frames per second, 80 frames per second, 90 frames per second,
100 frames per second, 120 frames per second, 150 frames per second
or 200 frames per second.
[0165] In some embodiments, the stage velocity fluctuation of the
optical scanning system is greater than +/-0.5%. In some
embodiments, the stage velocity fluctuation of the optical scanning
system is greater than +/-0.1%. In some embodiments, the stage
velocity fluctuation of the optical scanning system is greater than
+/-0.1%, and is reduced to less than +/-0.1% as observed by the
camera.
[0166] In some embodiments, the stage velocity fluctuation of the
optical scanning system is greater than +/-1%. In some embodiments,
the stage velocity fluctuation of the optical scanning system is
greater than +/-1%, and is reduced to less than +/-1% as observed
by the camera.
[0167] In some embodiments, the optical scanning system described
herein provides an increased sharpness of an image over a system
that does not compensate for velocity fluctuations in a
continuously moving stage.
[0168] In some embodiments, the total distance a substrate moves
during the imaging of a field deviates by more than +/-1 pixel (as
measured by the image of the substrate projected onto the sensor)
from a predetermined movement based on an anticipated velocity
during a capture of a field image, while the optical scanning
system generates an image with a pixel blur of less than 1. In some
embodiments, a pixel is correlated to an area of the field of--150
nm.times.150 nm. In some embodiments, a pixel is correlated to an
area of the field of--162.5 nm.times.162.5 nm. In some embodiments,
a pixel is correlated to an area of the field that is greater than
the size of a single fluorophore.
[0169] Pixel smear is one measure of image sharpness and refers to
an image artifact that results from the movement of a substrate in
an optical field with respect to an image sensor. One way to
measure pixel smear is by looking at the ratio of the major and
minor axes of a single spot, also known as the eccentricity. In
some embodiments, the eccentricity of an image generated by the
optical scanning system is less than 3. In some embodiments, the
eccentricity of the image is reliable single fluorophore
detection.
[0170] FIGS. 9A and 9B provides an example of pixel smear and
eccentricity of a resulting image of a substrate from the optical
scanning system provided herein. The blue spot represents a single
illuminated fluorophore, and each square is a pixel of--162 nm.
Shown in FIG. 9A is an example of a pixel smear of +1 pixel, within
a preferred range of +/-1 pixel, with an eccentricity of 2. Shown
in FIG. 9B is an example of a pixel smear of +2 pixels, outside of
the preferred range of +/-one pixel, with an eccentricity of 3.
[0171] FIG. 10 illustrates a system environment for transferring
information to or from the optical scanning device. The system
environment can include one or more client devices 1010, one or
more servers 1030, a database 1005 accessible to the server 1030,
where all of these parties are connected through a network 1020. In
other embodiments, different and/or additional entities can be
included in the system environment.
[0172] The system environment allows the results from the optical
scanning device 1040 to be shared via network 1020 with one or more
other users at their client devices 1010. Results can also be
uploaded to the web.
[0173] The network 1020 facilitates communications between the
components of the system environment. The network 1020 may be any
wired or wireless local area network (LAN) and/or wide area network
(WAN), such as an intranet, an extranet, or the Internet. In
various embodiments, the network 1020 uses standard communication
technologies and/or protocols. Examples of technologies used by the
network 1020 include Ethernet, 802.11, 3G, 4G, 802.16, or any other
suitable communication technology. The network 1020 may use
wireless, wired, or a combination of wireless and wired
communication technologies. Examples of networking protocols used
for communicating via the network 1020 include multiprotocol label
switching (MPLS), transmission control protocol/Internet protocol
(TCP/IP), hypertext transport protocol (HTTP), simple mail transfer
protocol (SMTP), and file transfer protocol (FTP). Data exchanged
over the network 1020 may be represented using any suitable format,
such as hypertext markup language (HTML) or extensible markup
language (XML). In some embodiments, all or some of the
communication links of the network 1020 may be encrypted using any
suitable technique or techniques.
[0174] The client device(s) 1010 are computing devices capable of
receiving user input as well as transmitting and/or receiving data
via the network 1020. In one embodiment, a client device 1010 is a
conventional computer system, such as a desktop or laptop computer.
Alternatively, a client device 1010 may be a device having computer
functionality, such as a personal digital assistant (PDA), a mobile
telephone, a smartphone or another suitable device. A client device
1010 is configured to communicate via the network 1020.
[0175] In some embodiments, the system environment may include one
or more servers, for example where the diagnostic system is
includes a service that is managed by an entity that communicates
via the network 1020 with the optical scanning device 1040 and/or
any of the client devices 1010. The server 1030 can store data in
database 1005 and can access stored data in database 1005. The
server 1030 may also store data in the cloud. In some embodiments,
the server 1030 may occasionally push updates to the optical
scanning device 1040, or may receive result data from the optical
scanning device 1040 and perform certain analyses on that result
data and provide the analyzed data back to the optical scanning
device 1040 or to a client device 1010.
[0176] In some embodiments, the optical scanning device 1040
functionality can be included in a client device 1010, such as a
mobile phone, and can be operated via a mobile application
installed on the phone. The mobile application stored on the phone
can process the results read from the optical scanning device and
share the results with other devices 810 on the network 820.
EQUIVALENTS AND SCOPE
[0177] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments in accordance with the
invention described herein. The scope of the present invention is
not intended to be limited to the above Description, but rather is
as set forth in the appended claims.
[0178] In the claims, articles such as "a," "an," and "the" may
mean one or more than one unless indicated to the contrary or
otherwise evident from the context. Claims or descriptions that
include "or" between one or more members of a group are considered
satisfied if one, more than one, or all of the group members are
present in, employed in, or otherwise relevant to a given product
or process unless indicated to the contrary or otherwise evident
from the context. The invention includes embodiments in which
exactly one member of the group is present in, employed in, or
otherwise relevant to a given product or process. The invention
includes embodiments in which more than one, or all of the group
members are present in, employed in, or otherwise relevant to a
given product or process.
[0179] It is also noted that the term "comprising" is intended to
be open and permits but does not require the inclusion of
additional elements or steps. When the term "comprising" is used
herein, the term "consisting of" is thus also encompassed and
disclosed.
[0180] Where ranges are given, endpoints are included. Furthermore,
it is to be understood that unless otherwise indicated or otherwise
evident from the context and understanding of one of ordinary skill
in the art, values that are expressed as ranges can assume any
specific value or subrange within the stated ranges in different
embodiments of the invention, to the tenth of the unit of the lower
limit of the range, unless the context clearly dictates
otherwise.
[0181] In addition, it is to be understood that any particular
embodiment of the present invention that falls within the prior art
may be explicitly excluded from any one or more of the claims.
Since such embodiments are deemed to be known to one of ordinary
skill in the art, they may be excluded even if the exclusion is not
set forth explicitly herein. Any particular embodiment of the
compositions of the invention (e.g., any nucleic acid or protein
encoded thereby; any method of production; any method of use; etc.)
can be excluded from any one or more claims, for any reason,
whether or not related to the existence of prior art.
[0182] All cited sources, for example, references, publications,
databases, database entries, and art cited herein, are incorporated
into this application by reference, even if not expressly stated in
the citation. In case of conflicting statements of a cited source
and the instant application, the statement in the instant
application shall control.
[0183] Section and table headings are not intended to be
limiting.
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