DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Priority
MPEP § 1895.01 states to “… require applicant to certify that the international application was not withdrawn or considered to be withdrawn, either generally or as to the United States, prior to the filing date of the national application claiming benefit under 35 U.S.C. 120 and 365(c) to such international application. In order to expedite examination, applicant should certify at the time of filing a national application claiming benefit under 35 U.S.C. 120 and 365(c) to an international application that the international application has not been withdrawn …”. Thus applicant is required to certify that the international application was not withdrawn or considered to be withdrawn, either generally or as to the United States, prior to the filing date of the national application claiming benefit under 35 U.S.C. 120 and 365(c) to such international application.
Drawings
The drawings were received on 13 April 2026. These drawings are acceptable.
Claim Interpretation
MPEP § 2111.01 states that “… Under a broadest reasonable interpretation (BRI), words of the claim must be given their plain meaning, unless such meaning is inconsistent with the specification. The plain meaning of a term means the ordinary and customary meaning given to the term by those of ordinary skill in the art at the relevant time. The ordinary and customary meaning of a term may be evidenced by a variety of sources, including the words of the claims themselves, the specification, drawings, and prior art. However, the best source for determining the meaning of a claim term is the specification - the greatest clarity is obtained when the specification serves as a glossary for the claim terms …”. Thus under a broadest reasonable interpretation, the greatest clarity is obtained when the specification (e.g., see “… With Atto 647 N fluorophore molecules having a fluorescence light emission peak with a maximum peak intensity at about 660 nm, the STED wavelength is at the far-red edge of the emission peak, where the remaining peak intensity is about 10% of the maximum peak intensity … STED wavelength may be set or at least successively reduced to a wavelength at which a fluorescence light emission peak of the singularized fluorophore molecule still has at least 25%, preferably at least 30%, and more preferably at least 35% of its maximum peak intensity … fluorescence inhibition effectiveness increases when the wavelength is successively blue-shifted from the red tail of the fluorophore's emission spectrum towards its emission maximum, which may allow for keeping the STED power constant …” in lines 15+ on pg. 3, lines 23+ on pg. 12, and lines 10+ on pg. 33) serves as a glossary for the claim term “the STED wavelength is set or successively reduced to a wavelength at which a fluorescence light emission peak of the singularized fluorophore molecule still has at least 25% of its maximum peak intensity”.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned at the time any inventions covered therein were effectively filed absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned at the time a later invention was effectively filed in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102 of this title, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claim(s) 1-4 is/are rejected under 35 U.S.C. 103 as being unpatentable over Hillman (US20160327779) in view of Schneider et al. (Ultrafast, temporally stochastic STED nanoscopy of millisecond dynamics, Nature Methods Vol. 12, no 9 (published online July 2015), pp. 827-830 and Supplementary Information) and Vicidomini et al. (STED with wavelengths closer to the emission maximum, Optics Express Vol. 20, no 5 (published February 2012), pp, 5225-5236).
In regard to claims 1 and 2, Hillman discloses a method of determining a molecule position of a singularized fluorophore molecule in an object, the method comprising:
(a) providing a light beam including fluorescence excitation light and fluorescence influencing light, wherein the fluorescence influencing light is STED light having a STED wavelength that is longer than an excitation wavelength of the fluorescence excitation light (e.g., “… illumination energy may suppress the image light at desired locations for resolution refinement … stimulated emission depletion (STED) imaging (e.g., by selectively deactivating fluorophores in specific regions while leaving a central focal spot active to emit fluorescence in the subject) … STED is a super-resolution technique that employs stimulated emission depletion to reduce the size of a diffraction limited spot or plane. In embodiments, STED can be achieved by aligning the secondary light source 124 to produce beams surrounding, bounding, or adjacent to the primary illumination. Thus, for point scanning, an annular beam spot of secondary illumination may be produced around the focal point of the primary illumination … STED is a super-resolution technique that employs stimulated emission depletion to reduce the diameter/width of the diffraction limited spot or sheet. In certain embodiments this can be achieved by aligning a second laser into a donut shape aligned around the focal point (for point scanning) … STED implementation can involve adding a second light source that is red-shifted from the one used for illumination and by way of a cylindrical lens, spatial light modulator or phase plate placed along its beam path, shape the beam into either a donut …” in paragraphs 151, 185, 188, and 506);
(b) shaping and focusing the light beam such as to form a light intensity distribution having a central intensity minimum of the STED light (e.g., “… STED implementation can involve adding a second light source that is red-shifted from the one used for illumination and by way of a cylindrical lens, spatial light modulator or phase plate placed along its beam path, shape the beam into … a donut …” in paragraph 506);
(c) continuously shifting the light intensity distribution with regard to the object such that the central intensity minimum is continuously moved along a track repeatedly extending around an estimated position of the singularized fluorophore molecule (e.g., “… scanning of the illumination beam may produce a continuous but complex or irregular pattern, such as circular, FIG. 8, Lissajous patterns, or even complex chaotic patterns, etc. With flexible control of scanning, as afforded by acousto-optical deflectors or SLMs and others, scanning may be controlled to cause the illumination beam to make more frequent visits on areas of greater interest, such as subject regions where more rapid motion is occurring or more complex features are present …” in paragraph 7);
(d) individually registering a plurality of individual photons of fluorescence light emitted by the singularized fluorophore molecule due to excitation by the fluorescence excitation light (e.g., “… imaging module 132 may include a linear or two-dimensional array of high-sensitivity detecting elements, such as photomultiplier tubes, avalanche photodiodes, or single-photon avalanche diodes. Alternatively or additionally, the imaging module 132 may include one or more waveguides (e.g., optical fibers) or conduits that direct light to a series of individual detectors or an array of detector elements …” in paragraph 178); and
(e) recording an intensity minimum position of the central intensity minimum for the excitation of each individual photon of the plurality of individual photons registered (e.g., “… control module 150 can correct for the real position of the illumination planar beam 137 within the subject. For example, the control module 150 could use feedback signals from the scanning 116 and the de-scanning 118 to determine actual angles and positions of the illuminated and detected light, as well as models of the optics and/or components of the system 100. Alternatively or additionally, the control module 150 can be configured to control system 100 to perform … stimulated emission depletion, or any other imaging modality …” in paragraph 181).
The method of Hillman lacks an explicit description of details of the “… scanning may be controlled to cause the illumination beam to make more frequent visits on areas of greater interest, such as subject regions where more rapid motion is occurring …” such as in response to each individual photon of the plurality of individual photons registered, updating the estimated position around which the track extends on basis of the recorded intensity minimum position, and at least one of reducing extensions of the track around the estimated position and increasing a fluorescence influencing effectiveness of the STED light and details of the “… STED implementation can involve adding a second light source that is red-shifted from the one used for illumination …” such as the STED wavelength is set or successively reduced to a wavelength at which a fluorescence light emission peak of the singularized fluorophore molecule still has at least 35% of its maximum peak intensity. However, “… scanning may be controlled …” details are known to one of ordinary skill in the art (e.g., see “… Pixel dwell times on the order of the fluorescence lifetime allowed the observation of dynamic processes with freely adjustable temporal resolution (as multiples of the ~1-ms frame time) … Smaller image fields would result in even higher frame rates … number of lines on the slow axis can be chosen flexibly … samples the detector signal, so it can assign the coordinate to each detected photon …” on pg. 829 and in the Optical setup section of Schneider et al.) and STED details are also known to one of ordinary skill in the art (e.g., see “… excited-state lifetime τ of a typical fluorophore is ~2-3 ns … synchronized the excitation laser at half of the repetition rate of the STED laser, i.e. fexc = fSTED/2 … gated STED (gSTED) implementation [18, 19, 24] by starting the Gate1 with a time-delay Tg from the excitation pulse … ATTO-647N emission spectra suggest a 2-3 fold increase of the stimulated cross-section when the STED wavelength is reduced from λSTED = 760 nm to λSTED = 730 nm… optimize the STED wavelength for a specific fluorophore, it also increases the number of suitable dyes for a given STED wavelength … desired fluorescent signal can be recovered if NFEx > √(NFEx +2NAStEx), or equivalently if the SNR = NFEx/√(NFEx + 2NAStEx) of the subtracted signal is > 1 …” on pp. 5228-5529 and 5233-5235 and “… (a,b) Experimental time sequence for … (b) P-STED implementations …” in the Fig. 1b caption of Vicidomini et al.). It should be noted that “when a patent claims a structure already known in the prior art that is altered by the mere substitution of one element for another known in the field, the combination must do more than yield a predictable results”. KSR International Co. v. Teleflex Inc., 550 U.S. 398 at 416, 82 USPQ2d 1385 (2007) at 1395 (citing United States v. Adams, 383 U.S. 39, 40 [148 USPQ 479] (1966)). See MPEP § 2143. In this case, one of ordinary skill in the art could have substituted a known conventional scanning (e.g., comprising details such as “number of lines on the slow axis can be chosen flexibly”, in order to achieve “even higher frame rates”) for the unspecified scanning of Hillman, could also have substituted a known conventional STED wavelength (e.g., comprising details such as “STED wavelength is reduced” so a “desired fluorescent signal can be recovered”, in order to “optimize the STED wavelength”) for the unspecified STED wavelength of Hillman, and the results of the substitutions would have been predictable. Therefore it would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to provide a known conventional STED scanning (e.g., comprising details such as in response to each individual photon of the plurality of individual photons registered, updating the estimated position around which the track extends on basis of the recorded intensity minimum position, and at least one of reducing extensions of the track around the estimated position and increasing a fluorescence influencing effectiveness of the STED light, wherein the STED wavelength is set or successively reduced to a wavelength at which a fluorescence light emission peak of the singularized fluorophore molecule still has at least 35% of its maximum peak intensity) as the unspecified STED scanning of Hillman.
In regard to claim 3 which is dependent on claim 1, the method of Hillman lacks an explicit description of details of the “… STED implementation can involve adding a second light source that is red-shifted from the one used for illumination …” such as at least one of notch- or edge-filtering the fluorescence light to suppress light of the STED wavelength prior to registering the plurality of individual photons and applying the STED light in pulses and gating the registering of the plurality of individual photons to select photons emitted after the respective pulse of the STED light. However, STED details are known to one of ordinary skill in the art (e.g., see “… excited-state lifetime τ of a typical fluorophore is ~2-3 ns … synchronized the excitation laser at half of the repetition rate of the STED laser, i.e. fexc = fSTED/2 … gated STED (gSTED) implementation [18, 19, 24] by starting the Gate1 with a time-delay Tg from the excitation pulse … ATTO-647N emission spectra suggest a 2-3 fold increase of the stimulated cross-section when the STED wavelength is reduced from λSTED = 760 nm to λSTED = 730 nm… optimize the STED wavelength for a specific fluorophore, it also increases the number of suitable dyes for a given STED wavelength … desired fluorescent signal can be recovered if NFEx > √(NFEx +2NAStEx), or equivalently if the SNR = NFEx/√(NFEx + 2NAStEx) of the subtracted signal is > 1 …” on pp. 5228-5529 and 5233-5235 and “… (a,b) Experimental time sequence for … (b) P-STED implementations …” in the Fig. 1b caption of Vicidomini et al.). It should be noted that “when a patent claims a structure already known in the prior art that is altered by the mere substitution of one element for another known in the field, the combination must do more than yield a predictable results”. KSR International Co. v. Teleflex Inc., 550 U.S. 398 at 416, 82 USPQ2d 1385 (2007) at 1395 (citing United States v. Adams, 383 U.S. 39, 40 [148 USPQ 479] (1966)). See MPEP § 2143. In this case, one of ordinary skill in the art could also have substituted a known conventional STED wavelength (e.g., comprising details such as “time sequence for … (b) P-STED implementations” so a “desired fluorescent signal can be recovered”, in order to “optimize the STED wavelength”) for the unspecified STED wavelength of Hillman and the results of the substitution would have been predictable. Therefore it would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to provide a known conventional STED scanning (e.g., comprising details such as applying the STED light in pulses and gating the registering of the plurality of individual photons to select photons emitted after the respective pulse of the STED light) as the unspecified STED scanning of Hillman.
In regard to claim 4 which is dependent on claim 1, the method of Hillman lacks an explicit description of details of the “… STED implementation can involve adding a second light source that is red-shifted from the one used for illumination …” such as the fluorescence influencing effectiveness of the fluorescence influencing light is increased by at least one of increasing an intensity of the STED light; increasing an effective fluorescence influencing cross section of the STED light by reducing its STED wavelength; and altering a temporal succession of pulses of the STED light and of pulses of the fluorescence excitation light. However, STED details are known to one of ordinary skill in the art (e.g., see “… excited-state lifetime τ of a typical fluorophore is ~2-3 ns … synchronized the excitation laser at half of the repetition rate of the STED laser, i.e. fexc = fSTED/2 … gated STED (gSTED) implementation [18, 19, 24] by starting the Gate1 with a time-delay Tg from the excitation pulse … ATTO-647N emission spectra suggest a 2-3 fold increase of the stimulated cross-section when the STED wavelength is reduced from λSTED = 760 nm to λSTED = 730 nm… optimize the STED wavelength for a specific fluorophore, it also increases the number of suitable dyes for a given STED wavelength … desired fluorescent signal can be recovered if NFEx > √(NFEx +2NAStEx), or equivalently if the SNR = NFEx/√(NFEx + 2NAStEx) of the subtracted signal is > 1 …” on pp. 5228-5529 and 5233-5235 and “… (a,b) Experimental time sequence for … (b) P-STED implementations …” in the Fig. 1b caption of Vicidomini et al.). It should be noted that “when a patent claims a structure already known in the prior art that is altered by the mere substitution of one element for another known in the field, the combination must do more than yield a predictable results”. KSR International Co. v. Teleflex Inc., 550 U.S. 398 at 416, 82 USPQ2d 1385 (2007) at 1395 (citing United States v. Adams, 383 U.S. 39, 40 [148 USPQ 479] (1966)). See MPEP § 2143. In this case, one of ordinary skill in the art could also have substituted a known conventional STED wavelength (e.g., comprising details such as “STED wavelength is reduced” so a “desired fluorescent signal can be recovered”, in order to “optimize the STED wavelength”) for the unspecified STED wavelength of Hillman and the results of the substitution would have been predictable. Therefore it would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to provide a known conventional STED scanning (e.g., comprising details such as the fluorescence influencing effectiveness of the fluorescence influencing light is increased by increasing an effective fluorescence influencing cross section of the STED light by reducing its STED wavelength) as the unspecified STED scanning of Hillman.
Claim(s) 5, 11, 12, 14-21, 23, and 24 is/are rejected under 35 U.S.C. 103 as being unpatentable over Hillman (US20160327779) in view of Schneider et al. (Ultrafast, temporally stochastic STED nanoscopy of millisecond dynamics, Nature Methods Vol. 12, no 9 (published online July 2015), pp. 827-830 and Supplementary Information).
In regard to claim 5, Hillman discloses a method of determining a molecule position of a singularized fluorophore molecule in an object, the method comprising:
(a) providing a light beam including fluorescence excitation light and fluorescence influencing light, wherein the fluorescence influencing light is identical to the fluorescence excitation light or wherein the fluorescence influencing light is fluorescence inhibition light provided in addition to the fluorescence excitation light (e.g., “… illumination energy may suppress the image light at desired locations for resolution refinement … stimulated emission depletion (STED) imaging (e.g., by selectively deactivating fluorophores in specific regions while leaving a central focal spot active to emit fluorescence in the subject) … STED is a super-resolution technique that employs stimulated emission depletion to reduce the size of a diffraction limited spot or plane. In embodiments, STED can be achieved by aligning the secondary light source 124 to produce beams surrounding, bounding, or adjacent to the primary illumination. Thus, for point scanning, an annular beam spot of secondary illumination may be produced around the focal point of the primary illumination … STED is a super-resolution technique that employs stimulated emission depletion to reduce the diameter/width of the diffraction limited spot or sheet. In certain embodiments this can be achieved by aligning a second laser into a donut shape aligned around the focal point (for point scanning) … STED implementation can involve adding a second light source that is red-shifted from the one used for illumination and by way of a cylindrical lens, spatial light modulator or phase plate placed along its beam path, shape the beam into either a donut …” in paragraphs 151, 185, 188, and 506);
(b) shaping and focusing the light beam such as to form a light intensity distribution having a central intensity minimum of the fluorescence influencing light (e.g., “… STED implementation can involve adding a second light source that is red-shifted from the one used for illumination and by way of a cylindrical lens, spatial light modulator or phase plate placed along its beam path, shape the beam into … a donut …” in paragraph 506);
(c) continuously shifting the light intensity distribution with regard to the object such that the central intensity minimum is continuously moved along a track repeatedly extending around an estimated position of the singularized fluorophore molecule (e.g., “… scanning of the illumination beam may produce a continuous but complex or irregular pattern, such as circular, FIG. 8, Lissajous patterns, or even complex chaotic patterns, etc. With flexible control of scanning, as afforded by acousto-optical deflectors or SLMs and others, scanning may be controlled to cause the illumination beam to make more frequent visits on areas of greater interest, such as subject regions where more rapid motion is occurring or more complex features are present …” in paragraph 7);
(d) individually registering a plurality of individual photons of fluorescence light emitted by the singularized fluorophore molecule due to excitation by the fluorescence excitation light (e.g., “… imaging module 132 may include a linear or two-dimensional array of high-sensitivity detecting elements, such as photomultiplier tubes, avalanche photodiodes, or single-photon avalanche diodes. Alternatively or additionally, the imaging module 132 may include one or more waveguides (e.g., optical fibers) or conduits that direct light to a series of individual detectors or an array of detector elements …” in paragraph 178); and
(e) recording an intensity minimum position of the central intensity minimum for the excitation of each individual photon of the plurality of individual photons registered (e.g., “… control module 150 can correct for the real position of the illumination planar beam 137 within the subject. For example, the control module 150 could use feedback signals from the scanning 116 and the de-scanning 118 to determine actual angles and positions of the illuminated and detected light, as well as models of the optics and/or components of the system 100. Alternatively or additionally, the control module 150 can be configured to control system 100 to perform … stimulated emission depletion, or any other imaging modality …” in paragraph 181).
The method of Hillman lacks an explicit description of details of the “… scanning may be controlled to cause the illumination beam to make more frequent visits on areas of greater interest, such as subject regions where more rapid motion is occurring …” such as in response to each individual photon of the plurality of individual photons registered, updating the estimated position around which the track extends on basis of the recorded intensity minimum position and at least one of reducing extensions of the track around the estimated position and increasing a fluorescence influencing effectiveness of the fluorescence influencing light. However, “… scanning may be controlled …” details are known to one of ordinary skill in the art (e.g., see “… Pixel dwell times on the order of the fluorescence lifetime allowed the observation of dynamic processes with freely adjustable temporal resolution (as multiples of the ~1-ms frame time) … Smaller image fields would result in even higher frame rates … number of lines on the slow axis can be chosen flexibly … samples the detector signal, so it can assign the coordinate to each detected photon …” on pg. 829 and in the Optical setup section of Schneider et al.). It should be noted that “when a patent claims a structure already known in the prior art that is altered by the mere substitution of one element for another known in the field, the combination must do more than yield a predictable results”. KSR International Co. v. Teleflex Inc., 550 U.S. 398 at 416, 82 USPQ2d 1385 (2007) at 1395 (citing United States v. Adams, 383 U.S. 39, 40 [148 USPQ 479] (1966)). See MPEP § 2143. In this case, one of ordinary skill in the art could have substituted a known conventional scanning (e.g., comprising details such as “number of lines on the slow axis can be chosen flexibly”, in order to achieve “even higher frame rates”) for the unspecified scanning of Hillman and the results of the substitution would have been predictable. Therefore it would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to provide a known conventional scanning (e.g., comprising details such as in response to each individual photon of the plurality of individual photons registered, updating the estimated position around which the track extends on basis of the recorded intensity minimum position and reducing extensions of the track around the estimated position) as the unspecified scanning of Hillman.
In regard to claim 11 which is dependent on claim 5, Hillman also discloses that the track extends around the estimated position of the singularized fluorophore molecule in all three spatial dimensions (e.g., “… illumination beam position and/or orientation is varied by one or more beam redirectors to support the formation of a two- or three-dimensional image … scanning of the illumination beam may produce a continuous but complex or irregular pattern, such as circular, FIG. 8, Lissajous patterns, or even complex chaotic patterns, etc. With flexible control of scanning, as afforded by acousto-optical deflectors or SLMs and others, scanning may be controlled to cause the illumination beam to make more frequent visits on areas of greater interest, such as subject regions where more rapid motion is occurring or more complex features are present …” in paragraphs 6 and 7).
In regard to claim 12 which is dependent on claim 5, the method of Hillman lacks an explicit description of details of the “… scanning may be controlled to cause the illumination beam to make more frequent visits on areas of greater interest, such as subject regions where more rapid motion is occurring …” such as the estimated position around which the track extends is updated on basis of the recorded intensity minimum position in that the estimated position is shifted away from the recorded intensity minimum position, if the fluorescence influencing light is identical to the fluorescence excitation light; or in that the estimated position is shifted towards the recorded intensity minimum position, if the fluorescence inhibition light is provided in addition to the fluorescence excitation light, by a predetermined distance fraction of a distance between the estimated position and the recorded intensity minimum position, wherein the distance fraction is in a range from 3% to 33%. However, “… scanning may be controlled …” details are known to one of ordinary skill in the art (e.g., see “… Pixel dwell times on the order of the fluorescence lifetime allowed the observation of dynamic processes with freely adjustable temporal resolution (as multiples of the ~1-ms frame time) … Smaller image fields would result in even higher frame rates … number of lines on the slow axis can be chosen flexibly … samples the detector signal, so it can assign the coordinate to each detected photon …” on pg. 829 and in the Optical setup section of Schneider et al.). It should be noted that “when a patent claims a structure already known in the prior art that is altered by the mere substitution of one element for another known in the field, the combination must do more than yield a predictable results”. KSR International Co. v. Teleflex Inc., 550 U.S. 398 at 416, 82 USPQ2d 1385 (2007) at 1395 (citing United States v. Adams, 383 U.S. 39, 40 [148 USPQ 479] (1966)). See MPEP § 2143. In this case, one of ordinary skill in the art could have substituted a known conventional scanning (e.g., comprising details such as “freely adjustable temporal resolution (as multiples of the ~1-ms frame time)”, in order to achieve “even higher frame rates”) for the unspecified scanning of Hillman and the results of the substitution would have been predictable. Therefore it would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to provide a known conventional scanning (e.g., comprising details such as the estimated position around which the track extends is updated on basis of the recorded intensity minimum position in that the estimated position is shifted away from the recorded intensity minimum position, if the fluorescence influencing light is identical to the fluorescence excitation light; or in that the estimated position is shifted towards the recorded intensity minimum position, if the fluorescence inhibition light is provided in addition to the fluorescence excitation light, by a predetermined distance fraction of a distance between the estimated position and the recorded intensity minimum position, wherein the distance fraction is in a range from 3% to 33%) as the unspecified scanning of Hillman.
In regard to claim 14 which is dependent on claim 5, the method of Hillman lacks an explicit description of details of the “… scanning may be controlled to cause the illumination beam to make more frequent visits on areas of greater interest, such as subject regions where more rapid motion is occurring …” such as the extensions of the track around the estimated position are reduced, unless predetermined minimum extensions have been reached, and the fluorescence influencing effectiveness of the fluorescence influencing light is increased, unless a predetermined maximum fluorescence influencing effectiveness has been reached. However, “… scanning may be controlled …” details are known to one of ordinary skill in the art (e.g., see “… Pixel dwell times on the order of the fluorescence lifetime allowed the observation of dynamic processes with freely adjustable temporal resolution (as multiples of the ~1-ms frame time) … Smaller image fields would result in even higher frame rates … number of lines on the slow axis can be chosen flexibly … samples the detector signal, so it can assign the coordinate to each detected photon …” on pg. 829 and in the Optical setup section of Schneider et al.). It should be noted that “when a patent claims a structure already known in the prior art that is altered by the mere substitution of one element for another known in the field, the combination must do more than yield a predictable results”. KSR International Co. v. Teleflex Inc., 550 U.S. 398 at 416, 82 USPQ2d 1385 (2007) at 1395 (citing United States v. Adams, 383 U.S. 39, 40 [148 USPQ 479] (1966)). See MPEP § 2143. In this case, one of ordinary skill in the art could have substituted a known conventional scanning (e.g., comprising details such as “freely adjustable temporal resolution”, in order to achieve “even higher frame rates”) for the unspecified scanning of Hillman and the results of the substitution would have been predictable. Therefore it would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to provide a known conventional scanning (e.g., comprising details such as the extensions of the track around the estimated position are reduced, unless predetermined minimum extensions have been reached, and the fluorescence influencing effectiveness of the fluorescence influencing light is increased, unless a predetermined maximum fluorescence influencing effectiveness has been reached) as the unspecified scanning of Hillman.
In regard to claim 15 which is dependent on claim 5, the method of Hillman lacks an explicit description of details of the “… scanning may be controlled to cause the illumination beam to make more frequent visits on areas of greater interest, such as subject regions where more rapid motion is occurring …” such as the movement of the central intensity minimum along the track is continued over the individual photons registered despite the updated estimated position and the reduced extensions of the track and the increased fluorescence influencing effectiveness of the fluorescence influencing light. However, “… scanning may be controlled …” details are known to one of ordinary skill in the art (e.g., see “… Pixel dwell times on the order of the fluorescence lifetime allowed the observation of dynamic processes with freely adjustable temporal resolution (as multiples of the ~1-ms frame time) … Smaller image fields would result in even higher frame rates … number of lines on the slow axis can be chosen flexibly … samples the detector signal, so it can assign the coordinate to each detected photon …” on pg. 829 and in the Optical setup section of Schneider et al.). It should be noted that “when a patent claims a structure already known in the prior art that is altered by the mere substitution of one element for another known in the field, the combination must do more than yield a predictable results”. KSR International Co. v. Teleflex Inc., 550 U.S. 398 at 416, 82 USPQ2d 1385 (2007) at 1395 (citing United States v. Adams, 383 U.S. 39, 40 [148 USPQ 479] (1966)). See MPEP § 2143. In this case, one of ordinary skill in the art could have substituted a known conventional scanning (e.g., comprising details such as “freely adjustable temporal resolution”, in order to achieve “even higher frame rates” for “the observation of dynamic processes”) for the unspecified scanning of Hillman and the results of the substitution would have been predictable. Therefore it would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to provide a known conventional scanning (e.g., comprising details such as despite the updated estimated position and the reduced extensions of the track and the increased fluorescence influencing effectiveness of the fluorescence influencing light, the movement of the central intensity minimum along the track is continued over the individual photons registered) as the unspecified scanning of Hillman.
In regard to claim 16 which is dependent on claim 5, the method of Hillman lacks an explicit description of details of the “… scanning may be controlled to cause the illumination beam to make more frequent visits on areas of greater interest, such as subject regions where more rapid motion is occurring …” such as a repetition rate at which the track extends around the estimated position of the singularized fluorophore molecule is at least 50% of a photon rate at which the individual photons are registered. However, “… scanning may be controlled …” details are known to one of ordinary skill in the art (e.g., see “… Pixel dwell times on the order of the fluorescence lifetime allowed the observation of dynamic processes with freely adjustable temporal resolution (as multiples of the ~1-ms frame time) … Smaller image fields would result in even higher frame rates … number of lines on the slow axis can be chosen flexibly … samples the detector signal, so it can assign the coordinate to each detected photon …” on pg. 829 and in the Optical setup section of Schneider et al.). It should be noted that “when a patent claims a structure already known in the prior art that is altered by the mere substitution of one element for another known in the field, the combination must do more than yield a predictable results”. KSR International Co. v. Teleflex Inc., 550 U.S. 398 at 416, 82 USPQ2d 1385 (2007) at 1395 (citing United States v. Adams, 383 U.S. 39, 40 [148 USPQ 479] (1966)). See MPEP § 2143. In this case, one of ordinary skill in the art could have substituted a known conventional scanning (e.g., comprising details such as “freely adjustable temporal resolution”, in order to achieve “even higher frame rates”) for the unspecified scanning of Hillman and the results of the substitution would have been predictable. Therefore it would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to provide a known conventional scanning (e.g., comprising details such as a repetition rate at which the track extends around the estimated position of the singularized fluorophore molecule is at least 50% of a photon rate at which the individual photons are registered) as the unspecified scanning of Hillman.
In regard to claim 17 which is dependent on claim 5, the method of Hillman lacks an explicit description of details of the “… scanning may be controlled to cause the illumination beam to make more frequent visits on areas of greater interest, such as subject regions where more rapid motion is occurring …” such as a signal indicating the individual photons registered is sampled at a sample rate that is at least 10 times a repetition rate at which the track extends around the estimated position of the singularized fluorophore molecule. However, “… scanning may be controlled …” details are known to one of ordinary skill in the art (e.g., see “… Pixel dwell times on the order of the fluorescence lifetime allowed the observation of dynamic processes with freely adjustable temporal resolution (as multiples of the ~1-ms frame time) … Smaller image fields would result in even higher frame rates … number of lines on the slow axis can be chosen flexibly … samples the detector signal, so it can assign the coordinate to each detected photon …” on pg. 829 and in the Optical setup section of Schneider et al.). It should be noted that “when a patent claims a structure already known in the prior art that is altered by the mere substitution of one element for another known in the field, the combination must do more than yield a predictable results”. KSR International Co. v. Teleflex Inc., 550 U.S. 398 at 416, 82 USPQ2d 1385 (2007) at 1395 (citing United States v. Adams, 383 U.S. 39, 40 [148 USPQ 479] (1966)). See MPEP § 2143. In this case, one of ordinary skill in the art could have substituted a known conventional scanning (e.g., comprising details such as “freely adjustable temporal resolution”, in order to achieve “even higher frame rates”) for the unspecified scanning of Hillman and the results of the substitution would have been predictable. Therefore it would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to provide a known conventional scanning (e.g., comprising details such as a signal indicating the individual photons registered is sampled at a sample rate that is at least 10 times a repetition rate at which the track extends around the estimated position of the singularized fluorophore molecule) as the unspecified scanning of Hillman.
In regard to claim 18 which is dependent on claim 5, Hillman also discloses that the track extends around the estimated position of the singularized fluorophore molecule in all three spatial dimensions, wherein the extensions of the track around the estimated position in the three spatial dimensions reflect extensions of an effective excitation point spread function of the light intensity distribution in the three spatial dimensions (e.g., “… illumination beam position and/or orientation is varied by one or more beam redirectors to support the formation of a two- or three-dimensional image … scanning of the illumination beam may produce a continuous but complex or irregular pattern, such as circular, FIG. 8, Lissajous patterns, or even complex chaotic patterns, etc. With flexible control of scanning, as afforded by acousto-optical deflectors or SLMs and others, scanning may be controlled to cause the illumination beam to make more frequent visits on areas of greater interest, such as subject regions where more rapid motion is occurring or more complex features are present …” in paragraphs 6 and 7).
In regard to claim 19 which is dependent on claim 18, Hillman also discloses that, in between the responses to two consecutive individual photons of the plurality of individual photons, the track runs on a surface of an ellipsoid having an x-half axis and y-half axis in a focal plane into which the light beam is focused and a z-half axis in a z-direction along which the light beam is focused into the focal plane, wherein the track revolves around the z-direction at an xy-revolution frequency which is not higher than an z-revolution frequency at which the track revolves around an axis rotating in the focal plane at the z-revolution frequency (e.g., “… illumination beam position and/or orientation is varied by one or more beam redirectors to support the formation of a two- or three-dimensional image … scanning of the illumination beam may produce a continuous but complex or irregular pattern, such as circular, FIG. 8, Lissajous patterns, or even complex chaotic patterns, etc. With flexible control of scanning, as afforded by acousto-optical deflectors or SLMs and others, scanning may be controlled to cause the illumination beam to make more frequent visits on areas of greater interest, such as subject regions where more rapid motion is occurring or more complex features are present …” in paragraphs 6 and 7). Alternatively it would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention that the “Lissajous patterns” of Hillman comprises xy-revolution frequency is not higher than an z-revolution frequency.
In regard to claim 20 which is dependent on claim 18, Hillman also discloses that, in between the responses to two consecutive individual photons of the plurality of individual photons, the track runs along a Lissajous curve, wherein the Lissajous curve passes through the estimated position (e.g., “… illumination beam position and/or orientation is varied by one or more beam redirectors to support the formation of a two- or three-dimensional image … scanning of the illumination beam may produce a continuous but complex or irregular pattern, such as circular, FIG. 8, Lissajous patterns, or even complex chaotic patterns, etc. With flexible control of scanning, as afforded by acousto-optical deflectors or SLMs and others, scanning may be controlled to cause the illumination beam to make more frequent visits on areas of greater interest, such as subject regions where more rapid motion is occurring or more complex features are present …” in paragraphs 6 and 7). Alternatively it would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention that the “Lissajous patterns” of Hillman comprises passing through the estimated position.
In regard to claim 21 which is dependent on claim 5, Hillman also discloses that, without any response to any photon registered, the track is a closed loop (e.g., “… scanning of the illumination beam may produce a continuous but complex or irregular pattern, such as circular, FIG. 8, Lissajous patterns, or even complex chaotic patterns, etc. With flexible control of scanning, as afforded by acousto-optical deflectors or SLMs and others, scanning may be controlled to cause the illumination beam to make more frequent visits on areas of greater interest, such as subject regions where more rapid motion is occurring or more complex features are present …” in paragraph 7).
In regard to claim 23 which is dependent on claim 5, Hillman also discloses that, once the molecule position of the singularized fluorophore molecule has been determined, another fluorophore molecule is singularized and a further molecule position of the other singularized fluorophore molecule in the object is determined (e.g., “… scanning of the illumination beam may produce a continuous but complex or irregular pattern, such as circular, FIG. 8, Lissajous patterns, or even complex chaotic patterns, etc. With flexible control of scanning, as afforded by acousto-optical deflectors or SLMs and others, scanning may be controlled to cause the illumination beam to make more frequent visits on areas of greater interest, such as subject regions where more rapid motion is occurring or more complex features are present …” in paragraph 7). Alternatively it would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention that the “areas of greater interest, such as subject regions where more rapid motion is occurring or more complex features are present” of Hillman comprises determining positions of at least two fluorophore molecules.
In regard to claim 24, Hillman discloses a laser-scanning microscope for determining a molecule position of a singularized fluorophore molecule in an object, the laser-scanning microscope comprising:
(a) a light source configured to provide a light beam including fluorescence excitation light and fluorescence influencing light, wherein the fluorescence influencing light is identical to the fluorescence excitation light or provided in addition to the fluorescence excitation light (e.g., “… illumination energy may suppress the image light at desired locations for resolution refinement … stimulated emission depletion (STED) imaging (e.g., by selectively deactivating fluorophores in specific regions while leaving a central focal spot active to emit fluorescence in the subject) … STED is a super-resolution technique that employs stimulated emission depletion to reduce the size of a diffraction limited spot or plane. In embodiments, STED can be achieved by aligning the secondary light source 124 to produce beams surrounding, bounding, or adjacent to the primary illumination. Thus, for point scanning, an annular beam spot of secondary illumination may be produced around the focal point of the primary illumination … STED is a super-resolution technique that employs stimulated emission depletion to reduce the diameter/width of the diffraction limited spot or sheet. In certain embodiments this can be achieved by aligning a second laser into a donut shape aligned around the focal point (for point scanning) … STED implementation can involve adding a second light source that is red-shifted from the one used for illumination and by way of a cylindrical lens, spatial light modulator or phase plate placed along its beam path, shape the beam into either a donut …” in paragraphs 151, 185, 188, and 506);
(b) a beam shaper and an objective configured to shape and focus the light beam such as to form a light intensity distribution having a central intensity minimum of the fluorescence influencing light (e.g., “… focusing module 108 (for example, an objective lenses or other reflective, diffractive, or refractive focusing optical components) receives the input illumination … STED implementation can involve adding a second light source that is red-shifted from the one used for illumination and by way of a cylindrical lens, spatial light modulator or phase plate placed along its beam path, shape the beam into … a donut …” in paragraphs 166 and 506);
(c) a scanner configured to continuously shift the light intensity distribution with regard to the object such that the central intensity minimum is continuously moved along a track repeatedly extending around an estimated position of the singularized molecule (e.g., “… scanning of the illumination beam may produce a continuous but complex or irregular pattern, such as circular, FIG. 8, Lissajous patterns, or even complex chaotic patterns, etc. With flexible control of scanning, as afforded by acousto-optical deflectors or SLMs and others, scanning may be controlled to cause the illumination beam to make more frequent visits on areas of greater interest, such as subject regions where more rapid motion is occurring or more complex features are present …” in paragraph 7);
(d) a detector configured to individually register a plurality of individual photons of fluorescence light emitted by the singularized molecule due to excitation by the fluorescence excitation light (e.g., “… imaging module 132 may include a linear or two-dimensional array of high-sensitivity detecting elements, such as photomultiplier tubes, avalanche photodiodes, or single-photon avalanche diodes. Alternatively or additionally, the imaging module 132 may include one or more waveguides (e.g., optical fibers) or conduits that direct light to a series of individual detectors or an array of detector elements …” in paragraph 178); and
(e) a controller configured to record an intensity minimum position of the central intensity minimum for the excitation of each individual photon of the plurality of individual photons registered (e.g., “… control module 150 can correct for the real position of the illumination planar beam 137 within the subject. For example, the control module 150 could use feedback signals from the scanning 116 and the de-scanning 118 to determine actual angles and positions of the illuminated and detected light, as well as models of the optics and/or components of the system 100. Alternatively or additionally, the control module 150 can be configured to control system 100 to perform … stimulated emission depletion, or any other imaging modality …” in paragraph 181).
The microscope of Hillman lacks an explicit description of details of the “… scanning may be controlled to cause the illumination beam to make more frequent visits on areas of greater interest, such as subject regions where more rapid motion is occurring …” such as the controller is further configured to, in response to each individual photon of the plurality of individual photons registered, update the estimated position around which the track extends on basis of the recorded intensity minimum position and at least one of reduce extensions of the track around the estimated position and increase a fluorescence influencing effectiveness of the fluorescence influencing light. However, “… scanning may be controlled …” details are known to one of ordinary skill in the art (e.g., see “… Pixel dwell times on the order of the fluorescence lifetime allowed the observation of dynamic processes with freely adjustable temporal resolution (as multiples of the ~1-ms frame time) … Smaller image fields would result in even higher frame rates … number of lines on the slow axis can be chosen flexibly … samples the detector signal, so it can assign the coordinate to each detected photon …” on pg. 829 and in the Optical setup section of Schneider et al.). It should be noted that “when a patent claims a structure already known in the prior art that is altered by the mere substitution of one element for another known in the field, the combination must do more than yield a predictable results”. KSR International Co. v. Teleflex Inc., 550 U.S. 398 at 416, 82 USPQ2d 1385 (2007) at 1395 (citing United States v. Adams, 383 U.S. 39, 40 [148 USPQ 479] (1966)). See MPEP § 2143. In this case, one of ordinary skill in the art could have substituted a known conventional scanning (e.g., comprising details such as “number of lines on the slow axis can be chosen flexibly”, in order to achieve “even higher frame rates”) for the unspecified scanning of Hillman and the results of the substitution would have been predictable. Therefore it would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to provide a known conventional scanning (e.g., comprising details such as the controller is further configured to, in response to each individual photon of the plurality of individual photons registered, update the estimated position around which the track extends on basis of the recorded intensity minimum position and reduce extensions of the track around the estimated position) as the unspecified scanning of Hillman.
Claim(s) 6-10 is/are rejected under 35 U.S.C. 103 as being unpatentable over Hillman in view of Schneider et al. as applied to claim(s) 5 above, and further in view of Vicidomini et al. (STED with wavelengths closer to the emission maximum, Optics Express Vol. 20, no 5 (published February 2012), pp, 5225-5236).
In regard to claim 6 which is dependent on claim 5, the method of Hillman lacks an explicit description of details of the “… STED implementation can involve adding a second light source that is red-shifted from the one used for illumination …” such as the fluorescence influencing effectiveness of the fluorescence influencing light is increased by at least one of increasing an intensity of the STED light; increasing an effective fluorescence influencing cross section of the STED light by reducing its STED wavelength; and altering a temporal succession of pulses of the STED light and of pulses of the fluorescence excitation light. However, STED details are known to one of ordinary skill in the art (e.g., see “… excited-state lifetime τ of a typical fluorophore is ~2-3 ns … synchronized the excitation laser at half of the repetition rate of the STED laser, i.e. fexc = fSTED/2 … gated STED (gSTED) implementation [18, 19, 24] by starting the Gate1 with a time-delay Tg from the excitation pulse … ATTO-647N emission spectra suggest a 2-3 fold increase of the stimulated cross-section when the STED wavelength is reduced from λSTED = 760 nm to λSTED = 730 nm… optimize the STED wavelength for a specific fluorophore, it also increases the number of suitable dyes for a given STED wavelength … desired fluorescent signal can be recovered if NFEx > √(NFEx +2NAStEx), or equivalently if the SNR = NFEx/√(NFEx + 2NAStEx) of the subtracted signal is > 1 …” on pp. 5228-5529 and 5233-5235 and “… (a,b) Experimental time sequence for … (b) P-STED implementations …” in the Fig. 1b caption of Vicidomini et al.). It should be noted that “when a patent claims a structure already known in the prior art that is altered by the mere substitution of one element for another known in the field, the combination must do more than yield a predictable results”. KSR International Co. v. Teleflex Inc., 550 U.S. 398 at 416, 82 USPQ2d 1385 (2007) at 1395 (citing United States v. Adams, 383 U.S. 39, 40 [148 USPQ 479] (1966)). See MPEP § 2143. In this case, one of ordinary skill in the art could also have substituted a known conventional STED wavelength (e.g., comprising details such as “STED wavelength is reduced” so a “desired fluorescent signal can be recovered”, in order to “optimize the STED wavelength”) for the unspecified STED wavelength of Hillman and the results of the substitution would have been predictable. Therefore it would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to provide a known conventional STED wavelength (e.g., comprising details such as the fluorescence influencing effectiveness of the fluorescence influencing light is increased by increasing an effective fluorescence influencing cross section of the STED light by reducing its STED wavelength) as the unspecified STED wavelength of Hillman.
In regard to claim 7 which is dependent on claim 5, the method of Hillman lacks an explicit description of details of the “… STED implementation can involve adding a second light source that is red-shifted from the one used for illumination …” such as the fluorescence influencing effectiveness of the fluorescence influencing light is increased by altering a temporal succession of pulses of the fluorescence influencing light and of pulses of the fluorescence excitation light. However, STED details are known to one of ordinary skill in the art (e.g., see “… excited-state lifetime τ of a typical fluorophore is ~2-3 ns … synchronized the excitation laser at half of the repetition rate of the STED laser, i.e. fexc = fSTED/2 … gated STED (gSTED) implementation [18, 19, 24] by starting the Gate1 with a time-delay Tg from the excitation pulse … ATTO-647N emission spectra suggest a 2-3 fold increase of the stimulated cross-section when the STED wavelength is reduced from λSTED = 760 nm to λSTED = 730 nm… optimize the STED wavelength for a specific fluorophore, it also increases the number of suitable dyes for a given STED wavelength … desired fluorescent signal can be recovered if NFEx > √(NFEx +2NAStEx), or equivalently if the SNR = NFEx/√(NFEx + 2NAStEx) of the subtracted signal is > 1 …” on pp. 5228-5529 and 5233-5235 and “… (a,b) Experimental time sequence for … (b) P-STED implementations …” in the Fig. 1b caption of Vicidomini et al.). It should be noted that “when a patent claims a structure already known in the prior art that is altered by the mere substitution of one element for another known in the field, the combination must do more than yield a predictable results”. KSR International Co. v. Teleflex Inc., 550 U.S. 398 at 416, 82 USPQ2d 1385 (2007) at 1395 (citing United States v. Adams, 383 U.S. 39, 40 [148 USPQ 479] (1966)). See MPEP § 2143. In this case, one of ordinary skill in the art could also have substituted a known conventional STED (e.g., comprising details such as “synchronized the excitation laser at half of the repetition rate of the STED laser”, in order to “optimize the STED wavelength”) for the unspecified STED of Hillman and the results of the substitution would have been predictable. Therefore it would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to provide a known conventional STED (e.g., comprising details such as the fluorescence influencing effectiveness of the fluorescence influencing light is increased by altering a temporal succession of pulses of the fluorescence influencing light and of pulses of the fluorescence excitation light) as the unspecified STED of Hillman.
In regard to claim 8 which is dependent on claim 7, the method of Hillman lacks an explicit description of details of the “… STED implementation can involve adding a second light source that is red-shifted from the one used for illumination …” such as intensities of the fluorescence influencing light and of the fluorescence excitation light are kept constant. However, STED details are known to one of ordinary skill in the art (e.g., see “… excited-state lifetime τ of a typical fluorophore is ~2-3 ns … synchronized the excitation laser at half of the repetition rate of the STED laser, i.e. fexc = fSTED/2 … gated STED (gSTED) implementation [18, 19, 24] by starting the Gate1 with a time-delay Tg from the excitation pulse … ATTO-647N emission spectra suggest a 2-3 fold increase of the stimulated cross-section when the STED wavelength is reduced from λSTED = 760 nm to λSTED = 730 nm… optimize the STED wavelength for a specific fluorophore, it also increases the number of suitable dyes for a given STED wavelength … desired fluorescent signal can be recovered if NFEx > √(NFEx +2NAStEx), or equivalently if the SNR = NFEx/√(NFEx + 2NAStEx) of the subtracted signal is > 1 …” on pp. 5228-5529 and 5233-5235 and “… (a,b) Experimental time sequence for … (b) P-STED implementations …” in the Fig. 1b caption of Vicidomini et al.). It should be noted that “when a patent claims a structure already known in the prior art that is altered by the mere substitution of one element for another known in the field, the combination must do more than yield a predictable results”. KSR International Co. v. Teleflex Inc., 550 U.S. 398 at 416, 82 USPQ2d 1385 (2007) at 1395 (citing United States v. Adams, 383 U.S. 39, 40 [148 USPQ 479] (1966)). See MPEP § 2143. In this case, one of ordinary skill in the art could also have substituted a known conventional STED (e.g., comprising details such as “synchronized the excitation laser at half of the repetition rate of the STED laser”, in order to “optimize the STED wavelength”) for the unspecified STED of Hillman and the results of the substitution would have been predictable. Therefore it would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to provide a known conventional STED (e.g., comprising details such as intensities of the fluorescence influencing light and of the fluorescence excitation light are kept constant) as the unspecified STED of Hillman.
In regard to claim 9 which is dependent on claim 7, the method of Hillman lacks an explicit description of details of the “… STED implementation can involve adding a second light source that is red-shifted from the one used for illumination …” such as the fluorescence influencing light is fluorescence inhibition light, wherein the fluorescence influencing light pulses are not longer than a fluorescence lifetime of the fluorophore molecule but longer than the excitation light pulses. However, STED details are known to one of ordinary skill in the art (e.g., see “… excited-state lifetime τ of a typical fluorophore is ~2-3 ns … synchronized the excitation laser at half of the repetition rate of the STED laser, i.e. fexc = fSTED/2 … gated STED (gSTED) implementation [18, 19, 24] by starting the Gate1 with a time-delay Tg from the excitation pulse … ATTO-647N emission spectra suggest a 2-3 fold increase of the stimulated cross-section when the STED wavelength is reduced from λSTED = 760 nm to λSTED = 730 nm… optimize the STED wavelength for a specific fluorophore, it also increases the number of suitable dyes for a given STED wavelength … desired fluorescent signal can be recovered if NFEx > √(NFEx +2NAStEx), or equivalently if the SNR = NFEx/√(NFEx + 2NAStEx) of the subtracted signal is > 1 …” on pp. 5228-5529 and 5233-5235 and “… (a,b) Experimental time sequence for … (b) P-STED implementations …” in the Fig. 1b caption of Vicidomini et al.). It should be noted that “when a patent claims a structure already known in the prior art that is altered by the mere substitution of one element for another known in the field, the combination must do more than yield a predictable results”. KSR International Co. v. Teleflex Inc., 550 U.S. 398 at 416, 82 USPQ2d 1385 (2007) at 1395 (citing United States v. Adams, 383 U.S. 39, 40 [148 USPQ 479] (1966)). See MPEP § 2143. In this case, one of ordinary skill in the art could also have substituted a known conventional STED (e.g., comprising details such as the Fig. 1b “time sequence”, in order to “optimize the STED wavelength”) for the unspecified STED of Hillman and the results of the substitution would have been predictable. Therefore it would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to provide a known conventional STED (e.g., comprising details such as the fluorescence influencing light is fluorescence inhibition light, wherein the fluorescence influencing light pulses are not longer than a fluorescence lifetime of the fluorophore molecule but longer than the excitation light pulses) as the unspecified STED of Hillman.
In regard to claim 10 which is dependent on claim 9, the method of Hillman lacks an explicit description of details of the “… STED implementation can involve adding a second light source that is red-shifted from the one used for illumination …” such as registering the photons of the fluorescence light is gated to select photons emitted after the respective fluorescence influencing light pulse and to block photons from the fluorophore molecule that did not yet experience the respective complete fluorescence influencing light pulse. However, STED details are known to one of ordinary skill in the art (e.g., see “… excited-state lifetime τ of a typical fluorophore is ~2-3 ns … synchronized the excitation laser at half of the repetition rate of the STED laser, i.e. fexc = fSTED/2 … gated STED (gSTED) implementation [18, 19, 24] by starting the Gate1 with a time-delay Tg from the excitation pulse … ATTO-647N emission spectra suggest a 2-3 fold increase of the stimulated cross-section when the STED wavelength is reduced from λSTED = 760 nm to λSTED = 730 nm… optimize the STED wavelength for a specific fluorophore, it also increases the number of suitable dyes for a given STED wavelength … desired fluorescent signal can be recovered if NFEx > √(NFEx +2NAStEx), or equivalently if the SNR = NFEx/√(NFEx + 2NAStEx) of the subtracted signal is > 1 …” on pp. 5228-5529 and 5233-5235 and “… (a,b) Experimental time sequence for … (b) P-STED implementations …” in the Fig. 1b caption of Vicidomini et al.). It should be noted that “when a patent claims a structure already known in the prior art that is altered by the mere substitution of one element for another known in the field, the combination must do more than yield a predictable results”. KSR International Co. v. Teleflex Inc., 550 U.S. 398 at 416, 82 USPQ2d 1385 (2007) at 1395 (citing United States v. Adams, 383 U.S. 39, 40 [148 USPQ 479] (1966)). See MPEP § 2143. In this case, one of ordinary skill in the art could also have substituted a known conventional STED (e.g., comprising details such as “gated STED (gSTED) implementation [18, 19, 24] by starting the Gate1 with a time-delay Tg from the excitation pulse”, in order to “optimize the STED wavelength”) for the unspecified STED of Hillman and the results of the substitution would have been predictable. Therefore it would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to provide a known conventional STED (e.g., comprising details such as registering the photons of the fluorescence light is gated to select photons emitted after the respective fluorescence influencing light pulse and to block photons from the fluorophore molecule that did not yet experience the respective complete fluorescence influencing light pulse) as the unspecified STED of Hillman.
Claim(s) 13 is/are rejected under 35 U.S.C. 103 as being unpatentable over Hillman in view of Schneider et al. as applied to claim(s) 5 above, and further in view of Tortarolo et al. (Synergic combination of stimulated emission depletion microscopy with image scanning microscopy to reduce light dosage (bioRxiv doi.org/10.1101/741389 (version posted August 2019), 18 pages).
In regard to claim 13 which is dependent on claim 5, the method of Hillman lacks an explicit description of details of the “… STED implementation can involve adding a second light source that is red-shifted from the one used for illumination …” such as the extensions of the track around the estimated position are reduced by an extensions fraction which is in a range from 1% to 10%, wherein the fluorescence influencing effectiveness of the fluorescence influencing light is increased such that extensions of an effective excitation point spread function of the light intensity distribution are also reduced by the extensions fraction. However, STED details are known to one of ordinary skill in the art (e.g., see “… the higher the STED beam intensity, the smaller the effective fluorescent region, and ultimately the better the spatial resolution … overall PSF of a STED microscope in the case of high STED intensities depends mainly on the excitation PSF, whilst the influence of the emission PSF becomes negligible … time-resolved STED [28] would potentially allow for a computationally efficient way to recombine photons originated from different regions of the effective STED fluorescent volume …” on pp. 2-4 of Tortarolo et al.). It should be noted that “when a patent claims a structure already known in the prior art that is altered by the mere substitution of one element for another known in the field, the combination must do more than yield a predictable results”. KSR International Co. v. Teleflex Inc., 550 U.S. 398 at 416, 82 USPQ2d 1385 (2007) at 1395 (citing United States v. Adams, 383 U.S. 39, 40 [148 USPQ 479] (1966)). See MPEP § 2143. In this case, one of ordinary skill in the art could also have substituted a known conventional STED (e.g., comprising details such as “overall PSF of a STED microscope in the case of high STED intensities depends mainly on the excitation PSF”, in order to achieve “the better the spatial resolution”) for the unspecified STED of Hillman and the results of the substitution would have been predictable. Therefore it would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to provide a known conventional STED (e.g., comprising details such as the extensions of the track around the estimated position are reduced by an extensions fraction which is in a range from 1% to 10%, wherein the fluorescence influencing effectiveness of the fluorescence influencing light is increased such that extensions of an effective excitation point spread function of the light intensity distribution are also reduced by the extensions fraction) as the unspecified STED of Hillman.
Claim(s) 22 is/are rejected under 35 U.S.C. 103 as being unpatentable over Hillman in view of Schneider et al. as applied to claim(s) 5 above, and further in view of Heine et al. (Adaptive-illumination STED nanoscopy, Proceedings of the National Academy of Sciences of the United States of America Vol. 114, no 37 (August 2017), pp. 9797-9802 and supporting Information).
In regard to claim 22 which is dependent on claim 5, the method of Hillman lacks an explicit description of details of the “… STED implementation can involve adding a second light source that is red-shifted from the one used for illumination …” such as prior to shaping and focusing the light beam such as to form the light intensity distribution having the central intensity minimum of the fluorescence influencing light, focusing a beam of the fluorescence excitation light such as to form an excitation-only light intensity distribution having a central intensity maximum; continuously shifting the excitation-only light intensity distribution with regard to the object; individually registering a preliminary plurality of individual photons of fluorescence light emitted by the singularized fluorophore molecule due to excitation by the fluorescence excitation light with a light detector array spatially resolving a distribution into which the fluorescence light emitted out of the central intensity maximum is imaged onto the light detector array; recording a detector array registration position and an intensity maximum position of the central intensity maximum for the excitation of each individual photon of the preliminary plurality of individual photons registered; and determining the estimated position to start with from the detector array registration positions and the intensity maximum positions recorded for the preliminary plurality of individual photons registered. However, STED details are known to one of ordinary skill in the art (e.g., see “… We start the scan away from the fluorophores without STED light, i.e., at confocal resolution. As the dye molecule is approached by the flank of the Gaussian excitation spot, the detected signal starts to increase, at which point the STED beam power is increased just enough that the fluorophore remains “off” given the new, enhanced resolution. At one of the next scan positions, the fluorophore emits again, and the STED power is further increased, and so on, until the desired STED power (i.e., resolution) is reached. Similarly, continuing the scan and moving away from the second fluorophore on the other side, the STED power is reduced to avoid scanning the crest across the fluorophore again. The fluorophores in DyMIN scanning therefore experience lower STED light doses than they would in a conventional scan, since the STED beam is not set to full power all of the time. Indeed, the highest power is applied at the scan positions centered at fluorophores and their vicinity …” on pg. 9798 of Heine et al.). It should be noted that “when a patent claims a structure already known in the prior art that is altered by the mere substitution of one element for another known in the field, the combination must do more than yield a predictable results”. KSR International Co. v. Teleflex Inc., 550 U.S. 398 at 416, 82 USPQ2d 1385 (2007) at 1395 (citing United States v. Adams, 383 U.S. 39, 40 [148 USPQ 479] (1966)). See MPEP § 2143. In this case, one of ordinary skill in the art could also have substituted a known conventional STED (e.g., comprising details such as “start the scan away from the fluorophores without STED light”, in order to “experience lower STED light doses than they would in a conventional scan, since the STED beam is not set to full power all of the time. Indeed, the highest power is applied at the scan positions centered at fluorophores and their vicinity”) for the unspecified STED of Hillman and the results of the substitution would have been predictable. Therefore it would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to provide a known conventional STED (e.g., comprising details such as prior to shaping and focusing the light beam such as to form the light intensity distribution having the central intensity minimum of the fluorescence influencing light, focusing a beam of the fluorescence excitation light such as to form an excitation-only light intensity distribution having a central intensity maximum; continuously shifting the excitation-only light intensity distribution with regard to the object; individually registering a preliminary plurality of individual photons of fluorescence light emitted by the singularized fluorophore molecule due to excitation by the fluorescence excitation light with a light detector array spatially resolving a distribution into which the fluorescence light emitted out of the central intensity maximum is imaged onto the light detector array; recording a detector array registration position and an intensity maximum position of the central intensity maximum for the excitation of each individual photon of the preliminary plurality of individual photons registered; and determining the estimated position to start with from the detector array registration positions and the intensity maximum positions recorded for the preliminary plurality of individual photons registered) as the unspecified STED of Hillman.
Response to Arguments
Applicant's arguments filed 13 April 2026 have been fully considered but they are not persuasive.
Applicant argues that interpretation under 35 U.S.C. § 112(f) on pg. 5 of the office action be withdrawn. Examiner respectfully disagrees. The previous office action stated “… MPEP § 2111.01 …” on pg. 5. MPEP § 2111.01 states that “… Under a broadest reasonable interpretation (BRI), words of the claim must be given their plain meaning, unless such meaning is inconsistent with the specification. The plain meaning of a term means the ordinary and customary meaning given to the term by those of ordinary skill in the art at the relevant time. The ordinary and customary meaning of a term may be evidenced by a variety of sources, including the words of the claims themselves, the specification, drawings, and prior art. However, the best source for determining the meaning of a claim term is the specification - the greatest clarity is obtained when the specification serves as a glossary for the claim terms …”. Thus under a broadest reasonable interpretation, the greatest clarity is obtained when the specification (e.g., see “… With Atto 647 N fluorophore molecules having a fluorescence light emission peak with a maximum peak intensity at about 660 nm, the STED wavelength is at the far-red edge of the emission peak, where the remaining peak intensity is about 10% of the maximum peak intensity … STED wavelength may be set or at least successively reduced to a wavelength at which a fluorescence light emission peak of the singularized fluorophore molecule still has at least 25%, preferably at least 30%, and more preferably at least 35% of its maximum peak intensity … fluorescence inhibition effectiveness increases when the wavelength is successively blue-shifted from the red tail of the fluorophore's emission spectrum towards its emission maximum, which may allow for keeping the STED power constant …” in lines 15+ on pg. 3, lines 23+ on pg. 12, and lines 10+ on pg. 33) serves as a glossary for the claim term “the STED wavelength is set or successively reduced to a wavelength at which a fluorescence light emission peak of the singularized fluorophore molecule still has at least 25% of its maximum peak intensity”.
Applicant argues that there is no citation from Hillman, and, in fact, the disclosure of Hillman does not refer to determining a molecule position of a singularized fluorophore molecule in an object. Examiner respectfully disagrees. As discussed in the previous office action, Hillman states (paragraphs 151, 185, 188, and 506) that “… illumination energy may suppress the image light at desired locations for resolution refinement … stimulated emission depletion (STED) imaging (e.g., by selectively deactivating fluorophores in specific regions while leaving a central focal spot active to emit fluorescence in the subject) … STED is a super-resolution technique that employs stimulated emission depletion to reduce the size of a diffraction limited spot or plane. In embodiments, STED can be achieved by aligning the secondary light source 124 to produce beams surrounding, bounding, or adjacent to the primary illumination. Thus, for point scanning, an annular beam spot of secondary illumination may be produced around the focal point of the primary illumination … STED is a super-resolution technique that employs stimulated emission depletion to reduce the diameter/width of the diffraction limited spot or sheet. In certain embodiments this can be achieved by aligning a second laser into a donut shape aligned around the focal point (for point scanning) … STED implementation can involve adding a second light source that is red-shifted from the one used for illumination and by way of a cylindrical lens, spatial light modulator or phase plate placed along its beam path, shape the beam into either a donut …”. It is important to recognize that “a central focal spot active to emit fluorescence in the subject” can be labeled as determining a molecule position and “selectively deactivating fluorophores in specific regions” can be labeled as singularized fluorophore molecule. Therefore, applicant's arguments are not persuasive.
Applicant argues that Hillman uses the light beam in a different way and for a different purpose than claimed. Examiner respectfully disagrees. As discussed in the previous office action, Hillman states (paragraph 7) that “… scanning of the illumination beam may produce a continuous but complex or irregular pattern, such as circular, FIG. 8, Lissajous patterns, or even complex chaotic patterns, etc. With flexible control of scanning, as afforded by acousto-optical deflectors or SLMs and others, scanning may be controlled to cause the illumination beam to make more frequent visits on areas of greater interest, such as subject regions where more rapid motion is occurring or more complex features are present …”. Thus Hillman teaches or suggests continuously (can be labeled “continuous but complex or irregular pattern”) shifting the light intensity distribution (can be labeled “scanning of the illumination beam”) with regard to the object such that the central intensity minimum is continuously moved along a track repeatedly extending around an estimated position (can be labeled “subject regions”) of the singularized fluorophore molecule “where more rapid motion is occurring or more complex features are present”. Therefore, the combination of the prior art teaches or suggests all limitations as arranged in the claims.
Applicant argues that the applicant’s Figs. 11 and 12 only very rarely visit the area of the estimated position of the singularized fluorophore molecule is a difference between the claimed invention and cited prior art. In response to applicant's argument that the references fail to show certain features of applicant’s invention, it is noted that the features upon which applicant relies (i.e., only very rarely visit the area of the estimated position of the singularized fluorophore molecule) are not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993).
Applicant argues that the present claims rely on the slope of the effective PSF, see e.g., Figs. 14A to 14C. In response to applicant's argument that the references fail to show certain features of applicant’s invention, it is noted that the features upon which applicant relies (i.e., the slope of the effective PSF) are not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993).
Applicant argues that Hillman does not assign any photons registered to a particular fluorophore molecule. In response to applicant's argument that the references fail to show certain features of applicant’s invention, it is noted that the features upon which applicant relies (i.e., assign any photons registered) are not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993).
Applicant argues that Hillman does not disclose individually registering individual photons of fluorescence light as recited in claim 1. Examiner respectfully disagrees. MPEP § 2111.01 states that “… Under a broadest reasonable interpretation (BRI), words of the claim must be given their plain meaning, unless such meaning is inconsistent with the specification. The plain meaning of a term means the ordinary and customary meaning given to the term by those of ordinary skill in the art at the relevant time. The ordinary and customary meaning of a term may be evidenced by a variety of sources, including the words of the claims themselves, the specification, drawings, and prior art. However, the best source for determining the meaning of a claim term is the specification - the greatest clarity is obtained when the specification serves as a glossary for the claim terms …”. Thus under a broadest reasonable interpretation, the greatest clarity is obtained when the specification (e.g., see “… detector 12 individually registers a plurality of individual photons of fluorescence light 40 emitted by the singularized molecule due to excitation by the fluorescence excitation light 4…” in lines 14+ on pg. 24) serves as a glossary for the claim term “registering a plurality of individual photons”. Further as discussed in the previous office action, Hillman states (paragraph 178) “… imaging module 132 may include a linear or two-dimensional array of high-sensitivity detecting elements, such as photomultiplier tubes, avalanche photodiodes, or single-photon avalanche diodes. Alternatively or additionally, the imaging module 132 may include one or more waveguides (e.g., optical fibers) or conduits that direct light to a series of individual detectors or an array of detector elements …”. Thus Hillman teaches or suggests using “single-photon avalanche diodes” for individually registering a plurality of individual photons of fluorescence light emitted by the singularized fluorophore molecule due to excitation by the fluorescence excitation light. Therefore, the combination of the prior art teaches or suggests all limitations as arranged in the claims.
Applicant argues that paragraph 181 nor any other paragraph of Hillman relates to an intensity minimum position of the central intensity minimum for excitation of each individual photon of the plurality of individual photons registered as recited in claim 1. Examiner respectfully disagrees. As discussed in the previous office action, Hillman states (paragraph 181) that “… control module 150 can correct for the real position of the illumination planar beam 137 within the subject. For example, the control module 150 could use feedback signals from the scanning 116 and the de-scanning 118 to determine actual angles and positions of the illuminated and detected light, as well as models of the optics and/or components of the system 100. Alternatively or additionally, the control module 150 can be configured to control system 100 to perform … stimulated emission depletion, or any other imaging modality …”. Thus Hillman teaches or suggests recording an intensity minimum position (can be labeled “determine actual angles and positions”) of the central intensity minimum for the excitation of each individual photon of the plurality of individual photons registered (can be labeled “detected light”). Therefore, the combination of the prior art teaches or suggests all limitations as arranged in the claims.
In response to applicant's argument that the examiner's conclusion of obviousness is based upon improper hindsight reasoning, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971).
In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
Applicant argues that none of the other cited prior art referred to in paragraph 13 of the office action teach or suggest at least the following basic principles of the present invention. Applicant's arguments fail to comply with 37 CFR 1.111(b) because they amount to a general allegation that the claims define a patentable invention without specifically pointing out how the language of the claims patentably distinguishes them from the references.
Applicant argues claims 2-4, which depend from claim 1, are not obvious for at least the same reasons as claim 1. Examiner respectfully disagrees for the reasons discussed above.
Applicant argues claims 6-10 depend from the non-obvious independent claims discussed above and are not obvious for at least the same reasons as the claims from which they depend. Examiner respectfully disagrees for the reasons discussed above.
Applicant argues claim 13 depend from a non-obvious independent claim discussed above and is not obvious for at least the same reasons as the claim from which it depends. Examiner respectfully disagrees for the reasons discussed above.
Applicant argues claim 22 depend from a non-obvious independent claim discussed above and is not obvious for at least the same reasons as the claim from which it depends. Examiner respectfully disagrees for the reasons discussed above.
Conclusion
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure.
US 20040212799 teaches STED.
US 20060038993 teaches STED.
US 20060044985 teaches STED.
US 20080007735 teaches STED.
US 20080018891 teaches STED.
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Shun Lee whose telephone number is (571)272-2439. The examiner can normally be reached Monday-Friday.
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/SL/
Examiner, Art Unit 2884
/UZMA ALAM/Supervisory Patent Examiner, Art Unit 2884
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