United States Patent [19]
`Hueton et al.
`
`I lllll llllllll Ill lllll lllll lllll lllll lllll lllll lllll lllll llllll Ill lllll llll
`US005459325A
`5,459,325
`Patent Number:
`Date of Patent:
`Oct. 17, 1995
`
`[45]
`
`[11]
`
`[54] HIGH-SPEED FLUORESCENCE SCANNER
`
`[75]
`
`Inventors: Iain Hueton, Los Altos Hills; Ezra
`Van Gelder, Belmont, both of Calif.
`
`[73] Assignee: Molecular Dynamics, Inc., Sunnyvale,
`Calif.
`
`[21] Appl. No.: 277,900
`
`Jul. 19, 1994
`
`[22] Filed:
`Int. Cl.6
`...........•.............................•........... GOIN 21/64
`[51]
`[52] U.S. Cl . ........................ 250/458.1; 356/317; 356/318
`[58] Field of Search .............................. 250/458.1, 461.1,
`250/461.2; 356/317, 318, 417
`
`[56]
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`4,684,797
`4,712,887
`4,877,966
`4,881,812
`5,001,694
`5,060,213
`5,088,079
`5,091,652
`5,195,074
`5,196,709
`
`8/1987 Ando et al .............................. 250/201
`12/1987 Baer ; ....................................... 350/484
`10/1989 Tomei et al. ......................... 250/458.1
`11/1989 Ohkubo et al .......................... 356/417
`3/1991 Lee et al .............................. 369/44.16
`10/1991 Karnisada ............................. 367/44.21
`2/1992 Baer ..................................... 369/44.26
`2/1992 Mathies et al. ...................... 250/458.1
`311993 Tanoshirna et al. ...................... 369/48
`311993 Berndt et al ......................... 250/458.1
`
`6/1993 Aoyarna et al ......................... 358/471
`5,218,461
`5,274,240 12/1993 Mathies et al ....................... 250/458.l
`5,293,363
`3/1994 Takeshita ............................. 369/44.21
`
`Primary Examiner-Carolyn E. Fields
`Attorney, Agent, or Firm-Schneck & McHugh
`
`[57]
`
`ABSTRACT
`
`A high-speed fluorescence scanner for scanning a sample at
`equal angles is disclosed. The scanner has most of its optical
`components, including a light beam source, a detector, and
`various filters, lenses, and reflectors, in a fixed position,
`removed from the scan head. The lightweight scan head
`contains a single reflector and lens combination which is
`reciprocated rapidly along one axis to lengthen and shorten
`a region of the path of a collimated excitation beam and to
`form a scan line on a sample. The fluorescence emission may
`be gathered by the lens of the scan head and directed back,
`generally along the optical path of the excitation beam, to a
`detector. Another embodiment of the scanner places the light
`source, in miniature form, directly on the scan head. The
`sample may be translated in an axis orthogonal to the scan
`line in order to stimulate fluorescent emission from a two(cid:173)
`dimensional portion of the sample. The design of the optical
`assembly currently permits scan speeds of up to approxi(cid:173)
`mately 100 inches per second.
`
`31 Claims, 5 Drawing Sheets
`
`THERMO FISHER EX. 1021
`
`

`
`U.S. Patent
`
`Oct. 17, 1995
`
`Sheet 1of5
`
`5,459,325
`
`('f")
`N
`
`0
`('f")
`
`('f")
`('f")
`
`0
`('f")
`
`~
`
`•
`CJ
`~
`~
`
`THERMO FISHER EX. 1021
`
`

`
`U.S. Patent
`
`Oct. 17, 1995
`
`Sheet 2 of 5
`
`5,459,325
`
`r
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`~
`0
`~ u
`~
`f--4
`~ a
`
`0\ -
`
`-
`
`-
`
`00 -
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`--,
`
`en
`en
`
`I'-... -
`
`\0 -
`- -~
`
`~ ~
`
`00 -
`
`00
`
`I.(\ -
`
`L - - - - - - - - - -
`
`_____ ...J
`
`-
`
`r
`I
`
`~ / N-
`
`0 .......
`
`0
`N
`
`THERMO FISHER EX. 1021
`
`

`
`U.S. Patent
`
`Oct. 17, 1995
`
`Sheet 3 of 5
`
`5,459,325
`
`20a
`!
`
`...
`
`30 33
`
`12
`
`FIG. 3A
`
`20b
`I
`
`30
`
`33
`
`12
`
`26
`
`25
`
`FIG. 3B
`
`THERMO FISHER EX. 1021
`
`

`
`U.S. Patent
`
`Oct. 17, 1995
`
`Sheet 4 of 5
`
`5,459,325
`
`20
`--~-----<--..
`
`30 33
`
`32a
`32b
`25
`
`FIG. 4
`
`THERMO FISHER EX. 1021
`
`

`
`01
`N
`~
`...
`\C
`01
`~
`...
`01
`
`(ll
`
`FIG. 5
`
`tl.l =-a
`
`(ll
`
`s,
`
`(ll
`~
`"""' ~
`"""' ~~
`:-"'
`t")
`0
`
`i
`
`""'d
`•
`rJJ.
`d •
`
`33
`
`17~
`
`23
`
`19-----iOETECTOR I
`
`33
`
`10
`
`37
`
`~
`
`(
`
`I(
`
`20
`
`THERMO FISHER EX. 1021
`
`

`
`1
`HIGH-SPEED FLUORESCENCE SCANNER
`
`5,459,325
`
`TECHNICAL FIELD
`This invention relates to optical scanners for stimulating 5
`and reading fluorescence emission from a target.
`
`BACKGROUND ART
`
`10
`
`15
`
`2
`directed to the scan head from a constant angle, and move(cid:173)
`ment of the scan head maintains a uniform angle of illumi(cid:173)
`nation on a sample or target. The effect of moving the scan
`head is continual lengthening and shortening of a region of
`the excitation beam's path to create a scan line in an image
`plane. Fluorescence emission from a sample placed within
`the image plane along the scan line of the excitation beam
`may be gathered by the lens of the scan head and directed
`back, generally along the optical path of the excitation beam,
`to a detector. The low mass of the scan head allows for high
`speed scanning. The scan head is driven by a linear actuator
`operable at up to approximately 100 inches per second. A
`sample may be moved orthogonally past the scan line for
`two-dimensional scanning, as by translation of a stage or by
`electrophoresis. The fluorescence scanner of the present
`invention may be adapted through substitution of lenses and
`reflectors' of various sizes and corresponding mechanical
`adjustments to provide for scanning and fluorescent imaging
`of samples in a wide variety of sizes and formats.
`Another embodiment of the present invention places the
`light source, in a miniature form, directly on the scan head.
`The scan head contains the light source and the lens. It may
`also contain a dichroic beam splitter or other means for
`separating the excitation beam from the fluorescence emis(cid:173)
`sion and for directing the fluorescence emission away from
`the scan head and toward a detector. In this alternate
`embodiment, there may be a fixed reflector, removed from
`the scan head, that is positioned to receive the fluorescence
`emission and deflect it toward the detector. Reciprocating
`motion of the scan head by the linear actuator in this instance
`30 causes a continual lengthening and shortening of the region
`of the fluorescence emission's path between the scan head
`and the reflector.
`The use of the term "reflector" includes mirrors and penta
`prisms. The use of the term "lens" signifies a single or
`35 multi-element lens.
`An advantage of the present invention over previous
`fluorescence scanners is the rapid stimulation of samples at
`a constant angle without resort to expensive corrective
`optics. In other words, the relative position of the scan head
`and the sample is maintained.
`Another advantage is the simple, lightweight design of the
`scan head and of the optical paths taken by the excitation
`beam and the fluorescence emission, which allow for a rapid
`scanning motion and for ease of adaptability to many
`applications.
`
`Fluorescence scanners generally scan samples via stimu(cid:173)
`lation by a light beam at an excitation wavelength, in a one
`or two-dimensional manner. The resulting stimulated fluo(cid:173)
`rescent emission, which typically occurs at a different wave(cid:173)
`length or wavelength band, is then detected. One type of
`scanner requires movement of an excitation beam in one
`axis and movement of a mechanical stage in an orthogonal
`axis so that successive straight line scans sequentially cover
`a two-dimensional area of the sample. Alternatively, the
`stage may be in a fixed position and the laser beam may be
`scanned along two axes. Also, the sample may be translated
`on an X-Y stage and viewed with a microscope or similar
`fixed optical viewer.
`The movement of a light beam to effect scanning in most
`fluorescence scanners is generally accomplished via galva(cid:173)
`nometer scanners and rotating polygonal mirrors. These
`devices are best suited for wide angle scanning, as is
`necessary for detecting fluorescence emission from planar
`DNA sequencing gels.
`It is important in certain applications to cause stimulation
`of fluorescence emission from a constant angle at all points
`of the specimen being scanned. There are inherent difficul(cid:173)
`ties in adapting the above scanning systems to such a
`situation because the scanning beams have some rotational
`motion and distortions of fluorescence imaging at various
`locations of the specimen may occur. Aberrations may be
`minimized through an f0 lens which, in conjunction with
`one of the above scanning mechanisms, provides correction
`of scan angle and speed and allows for scanning of a flat
`specimen with an incident beam. Such lenses are quite
`expensive, however. The costs of some of these scanning 40
`mechanisms is also very high.
`A wide variety of scan formats is necessary for many
`research and diagnostic applications. In particular, smaller
`experimental formats are emerging, such as the scanning of
`nucleic acid samples on small chips and electrophoresis
`within capillary tubes. Miniaturization of the effective scan-
`ning areas of existing fluorescent scanners requires intricate
`and expensive adaptation of optical assemblies and is,
`therefore, not feasible.
`It is also desirable to increase scan speed without com(cid:173)
`promising resolution in order to scan many samples in a
`short period of time. Existing scanners are limited with
`respect to scan speed and resolution because of their numer(cid:173)
`ous components and the high mass of their optical assem(cid:173)
`blies, and also because they are optimized to particular scan
`angles and sample sizes.
`It is therefore an object of the present invention to provide
`a versatile fluorescent scanner of simple, lightweight, low(cid:173)
`cost design for rapid scanning of a small format sample from
`a constant angle.
`
`20
`
`25
`
`45
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`FIG. 1 shows a scan head with accompanying support,
`and a portion of the optical assembly according to the
`present invention.
`FIG. 2 shows a plan view of the fluorescence scanner of
`the present invention, with optical assembly details.
`FIGS. 3A-B show a perspective view of a portion of the
`fluorescence scanner of the present invention, illustrating
`scan lines and two-dimensional fluorescence stimulation.
`FIG. 4 shows a perspective view of a portion of the
`fluorescence scanner of the present invention, illustrating
`translation in one dimension by electrophoresis.
`FIG. 5 shows an alternate embodiment of the present
`invention, with details of the scan head and optical assembly.
`
`BEST MODE OF CARRYING OUT THE
`INVENTION
`
`50
`
`55
`
`60
`
`DISCLOSURE OF THE INVENTION
`
`The above object has been achieved with a high-speed
`fluorescence scanner having rapid linear movement of a
`lightweight scan head containing only a single reflector and
`lens combination. The excitation beam is collimated and
`
`With reference to FIG. 1, a low-mass scan head 22 is
`65 shown to contain a reflector 13 and a lens 12. The use of the
`term "reflector" includes mirrors and penta prisms. The use
`of the term "lens" signifies a single or multi-element lens.
`
`THERMO FISHER EX. 1021
`
`

`
`5,459,325
`
`3
`Both reflector 13 and lens 12 are fixed in a given planar
`orientation within scan head 22, according to the desired
`optical scan angles. Lens 12 may be adjusted within the
`given planar orientation to achieve centration. Scan head 22
`may be attached to a bearing 21 which is slidably attached
`to a guide 27. In FIG. 1, guide 27 is shown as a U-shaped
`channel within which scan head 22 moves in direction 20.
`Linear actuator 23 is positioned at one end of guide 27 in the
`preferred embodiment and is in communication with scan
`head 22 through shaft 35, causing scan head 22 to recipro(cid:173)
`cate in direction 20 along guide 27, which shifts the overall
`position of reflector 13 and lens 12 and performs a scan in
`one dimension on image plane 10. Stop 37 is positioned at
`an end of guide 27 opposite linear actuator 23 and functions
`simply to keep bearing 21 on guide 27. Guide 27 may be
`mechanically supported at one or both opposing ends. The
`illustrated mechanical assembly shown is one example of a
`means of reciprocating scan head 22 linearly. Other means
`for rapid linear reciprocation may be substituted, such as an
`elongated arm having a movable member on which scan
`head 22 is mounted.
`With reference to FIG. 2, a complete diagram of one
`embodiment of the optical pathways is shown. The fluores(cid:173)
`cence scanner of the present invention generally has a fixed
`region 24 and a movable region defined by scan head 22.
`According to the preferred embodiment, within fixed region
`24, laser 11 emits a beam 30 at an excitation wavelength.
`Excitation beam 30 is collimated and optionally expanded
`by a pass through beam expander 14. A collimated white
`light source may also be used for excitation. A spectral
`dispersion device 16, such as a dichroic beam splitter, is
`placed within the path of excitation beam 30. Excitation
`beam 30 passes from spectral dispersion device 16 and then
`impinges upon a reflector 15 which is oriented at an approxi(cid:173)
`mately 45° angle to the incident excitation beam 30 in the
`preferred embodiment. Excitation beam 30 is then deflected
`in an orthogonal direction and moves away from fixed
`region 24 of FIG. 2 and enters scan head 22 by impinging
`upon reflector 13. The reflector 13 is also preferably placed
`at an approximately 45° angle to the incident excitation
`beam 30. Excitation beam 30 is then deflected in an orthogo(cid:173)
`nal direction by reflector 13. Lens 12, which generally has
`a clear aperture between one and twenty times greater than
`excitation beam 30, is positioned with its axis orthogonal to
`an image plane 10 so that it receives and focuses excitation
`beam 30 on image plane 10. Excitation beam 30 effects
`stimulation of fluorescent emission from samples or targets
`which are placed within image plane 10.
`Movement of scan head 22 in direction 20, as illustrated
`in FIGS. 1 and 2, causes a continual lengthening and
`shortening of the region of the collimated beam's path
`between reflectors 15 and 13 and results in scanning along
`one axis in image plane 10 while minimizing changes to the
`optical characteristics of beam 30 at image plane 10. The
`resulting fluorescence scanning resembles scanning by an
`optical disk read-head in preservation of beam properties.
`Motion of scan head 22 may be over long paths, such as
`several meters in length, or may be over short paths, such as
`less than one centimeter.
`FIGS. 3A-B more clearly illustrate the creation of scan
`lines. As the path of excitation beam 30 is lengthened
`between reflectors 15 and 13 by movement of scan head 22,
`in conjunction with linear actuator 23, in direction 20a, the
`combination of reflector 13 and lens 12 moves in direction
`20a to form scan line 25, as shown in FIG. 3A. In the same
`manner, as the path of excitation beam 30 is shortened by
`movement of scan head 22 in direction 20b, scan line 26 is
`
`15
`
`20
`
`4
`formed. Scan line 26 may be superimposed on scan line 25.
`If there is a shift of the sample within image plane 10 in a
`direction orthogonal to scan line 25, however, as indicated
`by arrow 29, then scan line 26 will be parallel to scan line
`5 25, as illustrated in FIG. 3B. Continual small shifts of the
`sample in direction 29 will cause formation of successive
`scan lines and result in two-dimensional fluorescent stimu(cid:173)
`lation of the sample in image plane 10.
`The movement of the sample in direction 29 may be
`10 accomplished by providing a stage for placement of the
`sample within image plane 10 and then by translation of the
`stage, as by a lead screw connected to a motor or another
`type of linear actuator. Alternatively, the sample may be
`translated in image plane 10 across the scan line by elec-
`trophoretic or other means. FIG. 4 gives an example of
`movement of a sample in one dimension by electrophoresis.
`Various components 32a-d of the sample are driven in
`direction 31 through a matrix 34 within lane 28 by appli(cid:173)
`cation of an electric field. Scan line 25 represents a scan in
`the direction 20 orthogonal to the direction 31 of sample
`movement. Continued electrophoretic movement of the
`sample components across the scan line allows for fluores(cid:173)
`cence detection of the entire sample over a period of time.
`For simplicity, FIG. 4 has been illustrated with only a few
`25 widely-spaced sample bands. The present invention is
`equally applicable, however, to fluorescent scanning of
`multiple closely-spaced sample bands and to multiple lanes
`of samples.
`Returning to FIG. 2, fluorescent emission by a sample
`30 placed within image plane 10 and scanned according to the
`present invention is gathered, in the preferred embodiment,
`by lens 12, which has a high numerical aperture and gathers
`fluorescence over a large angle. The term "high numerical
`aperture" means that the lens has a wide diameter for
`35 collected light, which is only fractionally used by the
`incoming beam, compared to its focal length. The gathered
`fluorescence is directed along a path substantially retracing
`the path of excitation beam 30. Fluorescence emission 33
`passes to reflector 13 and then to reflector 15. It is then
`40 directed to spectral dispersion device 16, which is positioned
`in the path of both the excitation beam 30 and the fluores(cid:173)
`cence emission 33, and operates to separate excitation from
`fluorescence emission via their unique spectral characteris(cid:173)
`tics. After the fluorescence emission 33 is separated by
`45 spectral dispersion device 16, it is passed through a lens 17
`and is then focused within an aperture 44 of a spatial filter
`18 and detected by a photosensitive detector 19, such as a
`photomultiplier tube or a photocell. The use of spatial filter
`18 is optional and is dependent upon the types of samples
`50 being scanned and the particular parameters of fluorescence
`detection necessary for such samples.
`By way of illustration, light sources for the present
`invention may include semiconductor or gas lasers, laser
`diodes, and may even be pulsed. A light emitting diode may
`55 also be used. FIG. 5 shows an alternate embodiment of the
`present invention, utilizing a miniature light source such as
`a laser diode, LED, halogen lamp, or xenon lamp. If the light
`source is in a miniature form, and therefore of low mass, it
`may be mounted directly on the scan head, instead of being
`in a fixed position removed from the scan head. Laser diode
`43 is shown in FIG. 5 as one example of a light source
`appropriate for the second embodiment. The excitation
`beam 41 emitted by laser diode 43 does not need to be
`collimated in this instance. Excitation beam 41 passes
`through a spectral dispersion device 40, such as a dichroic
`beam splitter, and then passes through lens 12. Lens 12
`focuses excitation beam 41 on the image plane to stimulate
`
`60
`
`65
`
`THERMO FISHER EX. 1021
`
`

`
`5,459,325
`
`5
`
`35
`
`5
`fluorescence emission. The resulting emission is generally
`gathered through the wide collection angle of lens 12 and
`then collimated. Fluorescence emission 33 then is directed
`out of scan head 22, preferably by spectral dispersion device
`40, which separates fluorescence emission 33 from excita-
`tion beam 41. Reflector 15 is placed in the path of fluores(cid:173)
`cence emission 33, preferably at an approximately 45°
`angle, and serves to deflect fluorescence emission 33 to lens
`17 for detection as in the first embodiment of the present
`invention. Reciprocation of scan head 22 by linear actuator
`23 causes scanning in one axis and the sample may be lO
`translated in another axis, as in the first embodiment. Fluo(cid:173)
`rescence emission 33 is thus transmitted along the axis of
`travel of the scan head.
`Within the optical assembly, the beam spot size and shape
`may be varied through the use of particular beam expanders
`and lenses. The depth of focus of the excitation beam is
`dependent upon the beam spot size and the focusing char(cid:173)
`acteristics of lens 12. Scanning may occur in a confocal or
`nonconfocal format in the present invention. In addition,
`fluorescence detection may occur in a manner that does not
`require retracing of the fluorescence emission along sub(cid:173)
`stantially the same optical path as that taken by the excita(cid:173)
`tion beam.
`Although beam paths parallel and orthogonal to the image
`plane have been illustrated for simplicity, positions of the
`optical components and the image plane may be altered
`depending on the application. For example, the direction
`from which excitation beam 30 impinges upon reflector 13
`and the orientations of reflector 13 and lens 12 within scan
`head 22 may be altered. Image plane 10 may also be shifted
`so that it is no longer parallel to the direction of linear
`motion of scan head 22 or so it receives excitation beam 30
`at all scan points from other than an orthogonal direction. As
`long as reflector 13 receives excitation beam 30 at a constant
`angle and the scan head components have been oriented to
`impinge upon a sample placed in an image plane, then linear
`reciprocation of the scan head in certain directions will
`cause scanning of the sample.
`The overall positions of the components of the fluores- 40
`cence scanner of the present invention may also be shifted.
`For example,. a sample may be mounted in a vertical image
`plane, the fixed region 24 may be positioned along a
`horizontal optical base plate, and the reciprocation mecha(cid:173)
`nism illustrated in FIG. 1 may be mounted on the base plate 45
`via a rigid bracket, so that linear actuator 23 causes scan
`head 22 to move vertically. This is the preferred positioning
`for scanning of samples such as vertical electrophoresis gels.
`According to the present invention, scan head 22 may
`reciprocate in a continual fashion and the laser scanner may 50
`operate for simple detection of fluorescent emission. For
`example, scan head 22 accelerates and reaches an optimum
`scan velocity at a known position along guide 27. In FIG. 1,
`sensor 38, which is positioned on guide 27, and flag 39,
`which is attached to scan head 22, operate in conjunction to 55
`mark the known home position of optimum scan velocity.
`Stimulation and reading of fluorescence then occurs for a
`period of time along the length of a scan line, after which the
`scan head decelerates and a new sample or line to be scanned
`may be placed within the scan line of excitation beam 30 60
`within image plane 10, e.g. via shifting of the sample in
`direction 29 of FIG. 3B. Successive, closely-spaced scans
`sequentially cover two dimensions in image plane 10. Alter(cid:173)
`natively, the laser scanner may be in communication with an
`image processing means whereby the scan mechanism col- 65
`lects fluorescence information from the sample in a location(cid:173)
`specific manner, as by determination of fluorescence inten-
`
`6
`sity within pixels approximating the beam spot size. This
`information may then be manipulated, e.g., to form a display
`of fluorescence locations. Reciprocation of scan head 22 in
`a continual, oscillating, or step-wise manner is anticipated.
`The scan head 22 and linear actuator 23 of the present
`invention generally operate in an open loop mode for data
`acquisition for image processing. For example, a command
`module may be attached to linear actuator 23 which sends
`scan head 22 out in one direction, as in direction 20a of FIG.
`3A, to a specified spot location, i.e. an address, then stops
`scan head 22 and sends it back in direction 20b of FIG. 3B.
`A closed loop mode may also be used depending on the
`accuracy required. The linear actuator may be of the type
`used in optical disk read-heads, except that the head may be
`15 supported at opposed ends, rather than being cantilevered or
`otherwise supported from just one end. The seek time to a
`single spot is preferably under 500 milliseconds, and more
`preferably under 50 milliseconds.
`The simple lightweight design and high scan speed of the
`20 present invention represent a significant advancement over
`the prior art. The scan head 22 is the only portion of the
`optical assembly that moves while scanning. The light beam
`source, detector, and various filters, lenses, and reflectors are
`in a fixed position, removed from scan head 22. The weight
`25 of scan head 22 is preferably under five hundred grams. The
`use of a miniature lens 12 and miniature reflector 13 further
`contributes to the light weight, and consequently, to the
`rapid scan speed. Linear actuator 23 is designed to operate
`in the range of 3 to 25 Hz and provide a scan speed of
`30 approximately 100 inches per second with current linear
`actuators. Examples of linear actuators appropriate to the
`present invention include voice coils, cams, belts, cranks, or
`lead screws connected to a motor to reciprocate scan head
`22, preferably in the axis represented by arrow 20. These
`linear actuators are preferably similar, in speed, accuracy
`and preservation of beam properties, to those used in optical
`disk read-heads.
`The on-axis configuration of the present invention also
`has the advantage of ensuring that excitation light impinges
`upon the sample at the same angle for all points of the
`sample in image plane 10. This is not the case with galva(cid:173)
`nometer scanners, polygonal mirrors, or raster systems. The
`present configuration allows for the use of less expensive
`lenses and eliminates aberrations associated with pin cush(cid:173)
`ion distortions, field curvature, and diffraction limiting
`lenses.
`The apparatus of the present invention may be used to
`detect the fluorescence of samples within gels, slides, dishes,
`capillary tubes, microtiter plates, cuvettes, or other formats
`in which high resolution, rapid scanning is necessary.
`Although a high-speed microscanner has been more particu(cid:173)
`larly described, the present invention may be configured to
`operate on larger samples. With the optical configuration of
`the present invention and minor adaptations to sizes and
`speeds of the various components, fluorescence scanning of
`samples in a wide variety of formats is possible.
`Simple adaptation to the present invention may also be
`made to allow for fluorescence detection in the Z-direction,
`or depthwise through the sample. This may be accom(cid:173)
`plished, e.g., through shifting of the sample stage to planes
`parallel to image plane 10 or by adding a movement of or
`within scan head 22 to cause a raising or lowering of the
`beam spot through the sample.
`Optical fibers to transmit the excitation beam or fluores(cid:173)
`cence emission along various portions of the optical path in
`the present invention may also be used.
`
`THERMO FISHER EX. 1021
`
`

`
`7
`
`5,459,325
`
`IO
`
`25
`
`We claim:
`1. A high-speed optical scanner for directing a beam at
`equal angles at a plurality of locations on a sample in an
`image plane comprising,
`means for producing a collimated excitation beam of light 5
`having a first spectral characteristic,
`a scan head having
`(i) a reflector disposed to receive the excitation beam at
`a constant angle and to deflect the excitation beam,
`and
`(ii) a lens disposed to intercept the excitation beam
`deflected by the reflector and to focus the excitation
`beam to a location on the sample in the image plane
`to cause fluorescent emission having a second spec(cid:173)
`tral characteristic from the sample, and to gather the 15
`fluorescent emission from the sample, the fluorescent
`emission being directed back to the reflector,
`actuator means for reciprocally moving the scan head in
`a linear direction, whereby
`the excitation beam
`impinges upon the sample within the image plane in a 20
`scan line,
`a spectral dispersion device for separating the spectral
`characteristics of fluorescent emission from the spectral
`characteristics of excitation.
`2. The scanner of claim 1 wherein the reflector receives
`the excitation beam from a direction parallel to the direction
`of motion of the means for reciprocally moving the scan
`head.
`3. The scanner of claim 2 wherein
`the reflector is oriented at an approximately 45° angle to
`the image plane, and
`the lens is positioned with its axis orthogonal to the image
`plane,
`whereby the excitation beam impinges upon the sample 35
`within the image plane from an orthogonal direction at
`all points in the scan line.
`4. The scanner of claim 1 wherein the actuator means is
`characterized by a scan speed of up to approximately 100
`inches per second.
`5. The scanner of claim 1 wherein the actuator means
`comprises a shaft mechanically coupled to the scan head, the
`shaft being driven axially by a motor.
`6. The scanner of claim 1 further comprising
`a means for translating the sample in the image plane in 45
`a direction orthogonal to the direction of the scan line,
`so that successive scans of the excitation beam sequen(cid:173)
`tially cover two dimensions in the image plane.
`7. The scanner of claim 1 further comprising an electro(cid:173)
`phoretic means for moving the sample in a direction 50
`orthogonal to the scan line.
`8. The scanner of claim 1 wherein the means for recip(cid:173)
`rocally moving the scan head comprises a linear actuator
`operating on an open loop mechanism.
`9. The scanner of claim i wherein the means for recipro(cid:173)
`cally moving the scan head comprises a linear actuator
`operating on a closed loop mechanism.
`10. The scanner of claim 1 wherein the means for pro(cid:173)
`ducing a collimated excitation beam comprises,
`a laser emitting an excitation beam, and
`a collimating lens positioned in the path of the excitation
`beam.
`11. The scanner of claim 1 further comprising,
`a second lens positioned proximate to the spectral disper- 65
`sion device, for focusing the fluorescent emission,
`which has been separated from the excitation beam by
`
`8
`the spectral dispersion device, and
`a light detector responsive to fluorescent emission.
`12. The scanner of claim 11 further comprising,
`a spatial filter positioned between the second lens and the
`light detector and having a pinhole aperture, the pin(cid:173)
`hole aperture disposed to intercept the fluorescence
`emission and to admit a portion thereof.
`13. A high-speed optical scanner for directing a beam at
`equal angles at a plurality of locations on a sample in an
`image plane comprising,
`a scan head having
`(i) means for producing an excitation beam of light
`having a first spectral characteristic,
`(ii) a lens disposed to intercept the excitation beam and
`to focus the excitation beam to a location on the
`sample in the image plane to cause fluorescent
`emission having a second spectral characteristic
`from the sample, and to gather the fluorescent emis(cid:173)
`sion from the sample,
`(iii) means for directing the fluorescent emission out of
`the scan head, and
`(iv) a spectral dispersion device for separating the
`spectral characteristics of fluorescent emission from
`the spectral characteristics of excitation, and
`actuator means for reciprocally moving the scan head in
`a linear direction, whereby
`the excitation beam
`impinges upon the sample within the image plane in a
`scan line.
`14. The scanner of claim 13 wherein the means for
`30 directing the fluorescent emission out of the scan head and
`the spectral dispersion device are a single dichroic beam
`splitter, the dichroic beam splitter positioned between the
`lens and the means for producing an excitation beam.
`15. The scanner of claim 13 wherein the means for
`producing an excitation beam is a light source selected from
`the group consisting of a laser diode, an LED, a halogen
`lamp, and a xenon lamp.
`16. The scanner of claim 13 wherein the actuator means
`is characterized by a scan speed of up to approximately 100
`inches per second.
`17. The scanner of claim 13 wherein the actuator means
`comprises a shaft mechanically coupled to the scan head, the
`shaft being driven axially by a motor.
`18. The scanner of claim 13 further comprising
`a means for translating the sample in the image plane in
`a direction orthogonal to the direction of the scan line,
`so that successive scans of the excitation beam sequen(cid:173)
`tially cover two dimensions in the image plane.
`19. The scanner of claim 13 further comprising an elec(cid:173)
`trophoretic means for moving the sample in a direction
`orthogonal to the scan line.
`20. The scanner of claim 13 wherein the means for
`reciprocally moving the scan head comprises a linear actua-
`tor operating on an open loop mechanism.
`21. The scanner of claim 13 wherein the means for
`reciprocally moving the scan head comprises a linear actua(cid:173)
`tor operating on a closed loop mechanism.
`22. The scanner of claim 13 further comprising,
`a light detector