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 .
Summary
This action is responsive to the amendments filed on 08/31/2025. The amendment has been entered. Applicant has submitted Claims 16 and 19-35 for examination. Applicant's amendments to the Specification, Drawings, and Claims have overcome each and every objection and 112(b) rejection previously set forth in the Non-Final Office Action mailed 02/10/2025.
Examiner finds the following: 1) Claims 16-35 are rejected; 2) no claims are objected to; and 3) no claims allowable.
Response to Arguments and Remarks
Examiner respectfully acknowledges Applicant's arguments, remarks, and amendments.
Regarding Applicant’s remarks about Balzarotti, Examiner is not persuaded.
Applicant notes that Balzarotti is generally directed to a miniflux and described as an alternative to STED. Examiner notes that both techniques are known in the art, used for this purpose, and follows similar principles. As such, Examiner is not persuaded.
Applicant’s remarks to saturation and Balzarotti are moot. Examiner relies on Wang for those limitations. As such, Examiner is not persuaded.
Applicant argues that Balzarotti fails to disclose a plurality of light distributions. Examiner, without agreeing with Applicant, notes that under MPEP 2144.04(VI)(B) – Duplication of Parts, that:
[T]he court held that mere duplication of parts has no patentable significance unless a new and unexpected result is produced.
Based on Examiner’s review and understanding, duplication of the light from Balzarotti would not produce new or unexpected results and that PHOSITA would have the skill to implement such duplications. As such, Examiner is not persuaded.
Regarding Applicant’s remarks about Wang, Examiner is not persuaded.
Applicant argues that the teaching of Wang are incompatible with Balzarotti. Examiner disagrees. Examiner relies on Wang for the teachings as it relates to saturation, not for the determining of position as Applicant argues. With regards to combining Balzarotti and Wang, Examiner is relying on Balrarotti as the cornerstone reference and notes how the saturation process is used and is known in a similar field such that PHOSITA would understand how to take the teachings of Wang to modify Balzarotti. As such, Examiner is not persuaded.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
Claims 16 and 19-35 are rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor regards as the invention.
Regarding Claims 16 and 33, both Claims 16 and 33 recite, in part:
… such that an effective detection point spread function is largely unaffected by the deactivation light distribution.
The most detail the specification goes into regarding this is while describing FIG. 1:
In particular, the method is performed such that the excitation light distribution 100 and the deactivation light distribution 300 during the different positionings of the excitation light distribution 100 are such that an effective detection PSF, in particular a width of the effective emission point spread function, is unaffected by the deactivation light distribution 300, i.e., is only affected by the excitation light distribution. This improves localization while suppressing background emission.
The only other two references to this in the specification appear to reiterate what is cited above.
It is unclear to Examiner what exactly “largely unaffected” means in the limitation. Due to the limited discussion of it in the specification, it is unclear if Applicant means to claim that the effective detection point function operates regardless of the deactivation light distribution, or perhaps that the effective point function operates in the background after the deactivation light distribution activates. Regardless, there is minimal support for what exactly “largely unaffected” means and as to how Examiner should interpret it. As such, Claims 16 and 33 are rejected as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor regards as the invention.
Regarding Claims 19-32 and 34-35, those claims depend on independent Claims 16 and 33, and as such, inherit the above issues.
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 (i.e., changing from AIA to pre-AIA ) 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.
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, 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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
Determining the scope and contents of the prior art.
Ascertaining the differences between the prior art and the claims at issue.
Resolving the level of ordinary skill in the pertinent art.
Considering objective evidence present in the application indicating obviousness or non-obviousness.
Claims 16, 24, and 33 are rejected under 35 U.S.C. 103 as being unpatentable over Balzarotti (Francisco Balzarotti et al. ,Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes.Science355,606-612(2017).DOI:10.1126/science.aak9913), in view of Wang (“Stimulated emission depletion microscopy with array detection and photon reassignment,” Wensheng Wang et. al., Optics and Lasers in Engineering, V. 129, June 2020, https://www.sciencedirect.com/science/article/pii/S014381661931797X).
Regarding Claim 16, Balzarotti discloses:
A method for determining positions of molecules spaced apart from one another in one or more spatial directions in a sample comprising:
- locating a single fluorophore (Balzarotti, P1, Abstract, “MINFLUX tracking of single fluorescent proteins”) and estimating the position of the fluorophore in a pre-localization (Balzarotti, P1, C2, “The emitter position is estimated from the centroid of the fluorescence diffraction pattern produced by the emitter on a camera (7). This process, called “localization,” can reach a precision given by the standard deviation of the diffraction fluorescence pattern divided by being the number of detected photons (8–11)”),
- generating a plurality of light distributions (Balzarotti, FIG. 1(C)), each light distribution comprising a local intensity minimum and intensity increasing regions adjacent thereto (Balzarotti, FIG. 1(A)), the light distributions comprising an excitation light distribution and a deactivation light distribution (Balzarotti, P1, C2, “The intensity and the wavelength of the pattern are adjusted such that molecular fluorescence is switched off (or on)”), particularly a STED light distribution (Balzarotti, P1, C1, “which most prominently include STED microscopy”),
- illuminating the sample with the excitation light distribution and the deactivation light distribution (Balzarotti, P3, C2, “λ = 405 nm illumination for on-switching, and λ = 642 nm excitation light”), wherein a local minimum of the excitation light distribution is successively arranged at a plurality of scanning positions within a scanning region near the position of the fluorophore that has been estimated in the pre-localization (Balzarotti, FIG. 1(C)),
- detecting photons emitted by a molecule for different positionings of the excitation light distribution (Balzarotti, FIG. 2(B)), wherein an emission signal is measured for each of the plurality of scanning positions, wherein the emission signal depends for each position of the excitation light distribution on a distance between the minimum of the excitation light distribution and the actual location of the fluorophore (Balzarotti, FIGS. 2(C-E)), wherein the closer to the minimum the fluorophore is located, the lower the emission signal (Balzarotti, shown at t2 in FIG. 2(D)), and
- deriving the position of the molecule separately from other molecules in the sample with increased accuracy compared to the pre-localization based on the photons detected for the different positionings of the excitation light distribution (Balzarotti, FIGS. 3(D-E),
wherein a light intensity of the deactivation … assigned to the scanning region (Balzarotti, FIG. 1(A), “The unknown position xm of a fluorescent molecule is determined by translating the standing wave, such that one of its intensity zeros travels from x =–L/2 to L/2,with xm being somewhere inbetween”), in which the position of the molecule can be unambiguously derived from the scanning positions and the associated detected photons, such that an effective detection point spread function is largely unaffected by the deactivation light distribution (Balzarotti, [0038], “Determining the positions of the individual fluorescent markers in the sample may particularly include fitting a spatial function having a local extremum to the intensities of the fluorescence light registered for the respective fluorescent marker for the different positions of the minimum”).
Balzarotti discloses the above, but does not explicitly disclose:
… light corresponds at most to a saturation intensity in a catch region …
However, Wang, in the similar field of endeavor (stimulated emission depletion microscopy), discloses:
… light corresponds at most to a saturation intensity in a catch region (Wang, FIG. 2(a)-2(f) and P2, C2, Paragraph 5, through P3, C1, Paragraph 1. Examiner notes that the effects of how saturation intensity is used to control the fluorescence observation value and altered spatial profile through point spread function, and thus the effect on the results, are known in the art) …
It would have been obvious to PHOSITA before the effective filing date of the claimed invention to modify Balzarotti with the resolving techniques of Wang. PHOSITA would have known about the uses of resolving techniques as disclosed by Wang and how to use them to modify the system of Balzarotti. PHOSITA would have been motivated to do this as a use of known technique to improve similar device in the same way (See MPEP § 2143 (I)(C)), specifically the use of known techniques to improve resolution using saturation for a STED apparatus.
Therefore, it would have been obvious to PHOSITA before Applicant’s filing date to have a light intensity to correspond to a saturation intensity, since the saturation intensity is a result effective variable and would require routine skill in the art. Furthermore, it has been held that that determining the optimum value of a result effective variable involves only routine skill in the art (see MPEP 2144.05 (II (A) and (B)).
Regarding Claim 24, the combination of Balzarotti and Wang discloses Claim 16, and Balzarotti further discloses:
…wherein the deactivation light distribution is formed as a 2D donut or a 3D donut (Balzarotti, FIG. 2(b)).
Regarding Claim 33, Balzarotti discloses:
A device, particularly a microscope, for determining positions of molecules spaced apart from one another in one or more spatial directions in a sample comprising
a. a pre-localization system comprising one or more pre-localization light sources configured to illuminate the sample with excitation light (Balzarotti, FIG. 1(C)), one or more pre-localization detectors configured to detect light from the sample (Balzarotti, FIG. 1(C)), and a pre-localization processor configured to locate a single fluorophore in the sample (Balzarotti, P1, Abstract, “MINFLUX tracking of single fluorescent proteins”) and estimate the position of the fluorophore from the detected light in a pre-localization (Balzarotti, P1, C2, “The emitter position is estimated from the centroid of the fluorescence diffraction pattern produced by the emitter on a camera (7). This process, called “localization,” can reach a precision given by the standard deviation of the diffraction fluorescence pattern divided by being the number of detected photons (8–11)”),
b. at least one light source identical to or different from the pre-localization light source, the at least one light source being configured to generate an excitation light beam and a deactivation light beam (Balzarotti, P1, C2, “The intensity and the wavelength of the pattern are adjusted such that molecular fluorescence is switched off (or on)”), particularly a STED beam (Balzarotti, P1, C1, “which most prominently include STED microscopy”),
c. at least one beam shaping device for forming an excitation light distribution from the excitation light beam and a deactivation light distribution from the deactivation light beam (Balzarotti, FIG. 2, Description of FIG. 2, “shaped by a vortex-phase mask”), wherein the excitation light distribution and the deactivation light distribution each comprise a local intensity minimum and intensity increasing regions adjacent thereto,
d.an optical arrangement for illuminating the sample with the excitation light distribution and the deactivation light distribution (Balzarotti, P1, C2, “The intensity and the wavelength of the pattern are adjusted such that molecular fluorescence is switched off (or on)”),
e. at least one beam deflection device configured to successively arrange the local minimum of the excitation light distribution at a plurality of scanning positions within a scanning region near the position of the fluorophore that has been estimated in the pre-localization (Balzarotti, FIG. 2(A), XY- Beam Deflector),
f. at least one detector identical to or different from the one or more pre-localization detectors (Balzarotti, FIG. 2(A), detector (DET)), the at least one detector being configured to detect for detecting photons emitted by a molecule for different positionings of the excitation light distribution (Balzarotti, FIGS. 2(A-E)), wherein the at least one detector is configured to measure an emission signal for each of the plurality of scanning positions (Balzarotti, FIGS. 2(A-E)), wherein the emission signal depends for each position of the excitation light distribution on a distance between the minimum of the excitation light distribution and the actual location of the fluorophore (Balzarotti, FIGS. 2(C-E)), wherein the closer to the minimum the fluorophore is located, the lower the emission signal (Balzarotti, shown at t2 in FIG. 2(D)), and
g. a Computing unit identical to or different from the pre-localization processor, the computing unit being configured to derive the position of the molecule separately from other molecules in the sample with increased accuracy compared to the pre-localization based on the photons detected for the different positionings of the excitation light distribution (Balzarotti, FIGS. 3(D-E), wherein the device comprises at least one beam deflection device configured to successively arrange the local minimum of the excitation light distribution at a plurality of scanning positions within a scanning region (Balzarotti, FIG. 2(A), XY- Beam Deflector), wherein the device comprises a control unit configured to adjust a light intensity of the deactivation light in a catch region assigned to the scanning region (Balzarotti, P6, C1, “Hence, adjusting L below the diffraction limit renders the emitted photons more informative. A perfect example is origami imaging (Fig. 4), where adjusting L from 70 nm to 50 nm improves the localization precision substantially. However, making L smaller must not be confused with exploiting external a priori information about molecular positions; no Bayesian estimation approach is needed. MINFLUX typically starts at the diffraction limit, but as soon as some position information is gained, L can be reduced and the uncertainty range “zoomed in.””), in which the position of the molecule can be unambiguously derived from the scanning positions and the associated detected photons, … such that an effective detection point spread function is largely unaffected by the deactivation light distribution (Balzarotti, [0038], “Determining the positions of the individual fluorescent markers in the sample may particularly include fitting a spatial function having a local extremum to the intensities of the fluorescence light registered for the respective fluorescent marker for the different positions of the minimum”).
Balzarotti discloses the above, but does not explicitly disclose:
… to at most three times a saturation intensity, …
However, Wang, in the similar field of endeavor (stimulated emission depletion microscopy), discloses:
… to at most three times a saturation intensity (Wang, FIG. 2(a)-2(f) and P2, C2, Paragraph 5, through P3, C1, Paragraph 1. Examiner notes that the effects of how saturation intensity is used to control the fluorescence observation value and altered spatial profile through point spread function, and thus the effect on the results, are known in the art), …
It would have been obvious to PHOSITA before the effective filing date of the claimed invention to modify Balzarotti with the resolving techniques of Wang. PHOSITA would have known about the uses of resolving techniques as disclosed by Wang and how to use them to modify the system of Balzarotti. PHOSITA would have been motivated to do this as a use of known technique to improve similar device in the same way (See MPEP § 2143 (I)(C)), specifically the use of known techniques to improve resolution using saturation for a STED apparatus.
Therefore, it would have been obvious to one having ordinary skill in the art before Applicant’s filing date to have a light intensity to correspond to a saturation intensity, since the saturation intensity is a result effective variable and would require routine skill in the art. Furthermore, it has been held that that determining the optimum value of a result effective variable involves only routine skill in the art (see MPEP 2144.05 (II (A) and (B)).
Claims 19-20, 22-23, 26-27, 29, 30, and 34-35 are rejected under 35 U.S.C. 103 as being unpatentable over Balzarotti (Francisco Balzarotti et al. ,Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes.Science355,606-612(2017).DOI:10.1126/science.aak9913), in view of Wang (“Stimulated emission depletion microscopy with array detection and photon reassignment,” Wensheng Wang et. al., Optics and Lasers in Engineering, V. 129, June 2020, https://www.sciencedirect.com/science/article/pii/S014381661931797X), and in further view of Tang (CN108181235A).
Regarding Claim 19, the combination of Balzarotti and Wang discloses Claim 16, but does not explicitly disclose:
…wherein the excitation light distribution is repositioned while the deactivation light distribution is left stationary so that the local minimum of the excitation light distribution is positioned differently more than once within a vicinity of the local intensity minimum of the deactivation light distribution.
However, Tang, in a similar field of endeavor (Uniform Structured Light Illumination Based STED Parallel Micro-imaging System, Has Image Rebuilding Module For Performing Image Reconstruction By Using SIM Frequency Domain Spectrum Map To Realize STED Microscopic Imaging Process), discloses:
…wherein the excitation light distribution is repositioned while the deactivation light distribution is left stationary so that the local minimum of the excitation light distribution is positioned differently more than once within a vicinity of the local intensity minimum of the deactivation light distribution (Tang, FIG. 1 and P8, Paragraph 6, “The Wollaston prism 114 is rotated, and the phase map loaded on the liquid crystal spatial light modulator 115 is rotated accordingly, so that the structured light field fringes of the excitation light and the loss light at the focal plane of the objective lens 1123 can be located in different directions to generate different directions.” Examiner notes that prisms 113 and 114 are independent of each other, such that the loss light distribution can be stationary relative to the light distribution as claimed, which would allow for the modification of one light while keeping the other unmodified).
It would have been obvious to PHOSITA before the effective filing date of the claimed invention to modify the combination of Balzarotti and Wang with the stationary deactivation light of Tang. PHOSITA would have known about the uses of stationary deactivation light as disclosed by Tang and how to use them to modify the combination of Balzarotti and Wang. PHOSITA would have been motivated to do this as a use of known technique to improve similar device in the same way (See MPEP § 2143 (I)(C)), specifically the use a stationary deactivation light to generate different directions and angles of light in conjunction with the activation light.
Regarding Claim 20, the combination of Balzarotti and Wang discloses Claim 16, but does not explicitly disclose:
…wherein the excitation light distribution and the deactivation light distribution are repositioned together.
However, Tang, in a similar field of endeavor (Uniform Structured Light Illumination Based STED Parallel Micro-imaging System, Has Image Rebuilding Module For Performing Image Reconstruction By Using SIM Frequency Domain Spectrum Map To Realize STED Microscopic Imaging Process), discloses:
…wherein the excitation light distribution and the deactivation light distribution are repositioned together (Tang, FIG. 1 and P8, Paragraph 4, “The structure light (excitation structure light and loss structure light) is maintained in the same direction, and the three-dimensional nano-movement stage 1115 is used to drive the sample to move step by step in a direction perpendicular to the structure light stripe.” Examiner notes that that in Tang that the stage is moving in the above, but that is a matter of relative perspective. The excitation structure light and loss structure light are moving together, as one, relative to the stage for the repositioning).
It would have been obvious to PHOSITA before the effective filing date of the claimed invention to modify the combination of Balzarotti and Wang with the mobile deactivation light of Tang. PHOSITA would have known about the uses of mobile deactivation light as disclosed by Tang and how to use them to modify the combination of Balzarotti and Wang. PHOSITA would have been motivated to do this as a use of known technique to improve similar device in the same way (See MPEP § 2143 (I)(C)), specifically the use a mobile deactivation light to generate consistent directions and angles of light in conjunction with the activation light.
Regarding Claim 22, the combination of Balzarotti and Wang discloses Claim 16, and Balzarotti generally discloses scanning throughout but does not explicitly disclose:
…wherein a plurality of scanning steps is performed, wherein the local minimum of the excitation light distribution in each of the scanning steps is positioned at a plurality of the scanning positions of a respective scanning region, wherein the respective scanning region of each scanning step comprises a smaller area or volume than the scanning regions of the preceding scanning steps, and the deactivation light distribution is adjusted in at least a part of the scanning steps depending on the area or volume of the respective scanning area, wherein a total intensity and/or a shape of the deactivation light distribution is adjusted depending on the area or volume of the respective scanning area.
However, Tang, in a similar field of endeavor (Uniform Structured Light Illumination Based STED Parallel Micro-imaging System, Has Image Rebuilding Module For Performing Image Reconstruction By Using SIM Frequency Domain Spectrum Map To Realize STED Microscopic Imaging Process), discloses:
…wherein a plurality of scanning steps is performed, wherein the local minimum of the excitation light distribution in each of the scanning steps is positioned at a plurality of the scanning positions of a respective scanning region (Tang, FIG. 1 and P8, Paragraph 4, “The structure light (excitation structure light and loss structure light) is maintained in the same direction, and the three-dimensional nano-movement stage 1115 is used to drive the sample to move step by step in a direction perpendicular to the structure light stripe”), wherein the respective scanning region of each scanning step comprises a smaller area or volume than the scanning regions of the preceding scanning steps (Tang, FIG. 1 and P8, Paragraph 4, “The total number of movements in the direction of each structure light stripe is dp/ Ds-1,dp is the fringe space period, ds is the single movement step, each position of the displacement stage stays one position, the area array detector 123 is used to record the sample stripe fluorescence image at the position, and the second step is adopted.” Examiner notes that Tang’s set up would allow for various scanning region sizes), and the deactivation light distribution is adjusted in at least a part of the scanning steps depending on the area or volume of the respective scanning area, wherein a total intensity and/or a shape of the deactivation light distribution is adjusted depending on the area or volume of the respective scanning area (Tang, FIG. 1 and P8, Paragraph 4, “The total number of movements in the direction of each structure light stripe is dp/ Ds-1,dp is the fringe space period, ds is the single movement step, each position of the displacement stage stays one position, the area array detector 123 is used to record the sample stripe fluorescence image at the position, and the second step is adopted.” Examiner notes that Tang’s set up would allow for various scanning region sizes.” Examiner notes that it would not make sense for Tang’s operation to not be adjustable depending on the size and area of scanning).
It would have been obvious to PHOSITA before the effective filing date of the claimed invention to modify the combination of Balzarotti and Wang with the scanning techniques of Tang. PHOSITA would have known about the scanning techniques as disclosed by Tang and how to use them to modify the combination of Balzarotti and Wang. PHOSITA would have been motivated to do this as a use of known technique to improve similar device in the same way (See MPEP § 2143 (I)(C)), specifically the use known scanning techniques in STED systems.
Regarding Claim 23, the combination of Balzarotti and Wang discloses Claim 16, but does not explicitly disclose:
…wherein the deactivation light distribution remains constant during repositioning of the excitation light distribution.
However, Tang, in a similar field of endeavor (Uniform Structured Light Illumination Based STED Parallel Micro-imaging System, Has Image Rebuilding Module For Performing Image Reconstruction By Using SIM Frequency Domain Spectrum Map To Realize STED Microscopic Imaging Process), discloses:
…wherein the deactivation light distribution remains constant during repositioning of the excitation light distribution (Tang, FIG. 1 and P8, Paragraph 6, “The Wollaston prism 114 is rotated, and the phase map loaded on the liquid crystal spatial light modulator 115 is rotated accordingly, so that the structured light field fringes of the excitation light and the loss light at the focal plane of the objective lens 1123 can be located in different directions to generate different directions.” Examiner notes that prisms 113 and 114 are independent of each other, such that the loss light distribution can be stationary relative to the light distribution as claimed, which would allow for the modification of one light while keeping the other unmodified).
It would have been obvious to PHOSITA before the effective filing date of the claimed invention to modify the combination of Balzarotti and Wang with the constant deactivation light distribution of Tang. PHOSITA would have known about the uses of constant deactivation light distribution as disclosed by Tang and how to use them to modify the combination of Balzarotti and Wang. PHOSITA would have been motivated to do this as a use of known technique to improve similar device in the same way (See MPEP § 2143 (I)(C)), specifically the use a mobile deactivation light with constant deactivation light distribution to generate consistent directions and angles of light in conjunction with the activation light.
Regarding Claim 26, the combination of Balzarotti and Wang discloses Claim 16, but does not explicitly disclose:
…wherein the deactivation light distribution is formed by irradiating a deactivation light beam onto the sample along an optical axis.
Balzarotti hints at such a system in FIG. 2A, but not with the specificity Examiner was looking for.
However, Tang, in a similar field of endeavor (Uniform Structured Light Illumination Based STED Parallel Micro-imaging System, Has Image Rebuilding Module For Performing Image Reconstruction By Using SIM Frequency Domain Spectrum Map To Realize STED Microscopic Imaging Process), discloses:
…wherein the deactivation light distribution is formed by irradiating a deactivation light beam onto the sample along an optical axis (Tang, FIG. 1 and P6, Paragraph 3, “The mirror 1121, the first dichroic mirror 1116, the filter 121, and the detection lens 122 form an even striped pattern image on the photosensitive surface of the area array detector 123), wherein the deactivation light beam is focused into the sample by an objective (Tang, FIG. 1 and P6, Paragraph 2, “After the wave plate 1119 has the same polarization direction, the first loss light and the second loss light after passing through the second half wave plate 1119 enter the objective lens after passing through the second dichroic mirror 1121 and the barrel mirror 1122 in sequence”), and wherein a beam cross-section of the deactivation light beam is adjusted such that a pupil of the objective lens is under-illuminated so that the deactivation light distribution is stretched in the direction of the optical axis (Tang, FIG. 1 and P6, Paragraph 2, “After the wave plate 1119 has the same polarization direction, the first loss light and the second loss light after passing through the second half wave plate 1119 enter the objective lens after passing through the second dichroic mirror 1121 and the barrel mirror 1122 in sequence. 1123, and perform interference at the front focal plane of the objective lens 1123 to generate a uniform distribution of interference fringe-like loss structure light, and the loss structure light depletes the sample”).
It would have been obvious to PHOSITA before the effective filing date of the claimed invention to modify the combination of Balzarotti and Wang with the axis considerations of Tang. PHOSITA would have known about the axis considerations as disclosed by Tang and how to use them to modify the combination of Balzarotti and Wang. PHOSITA would have been motivated to do this as a use of known technique to improve similar device in the same way (See MPEP § 2143 (I)(C)), specifically the use a common axis and control for uniform distribution of the light pattern.
Regarding Claim 27, the combination of Balzarotti, Wang, and Tang discloses Claim 26, and Tang further discloses:
…wherein the beam cross-section of the deactivation light beam is adjusted by adapting an active surface of a beam shaping device, particularly a light modulator (Tang, FIG. 1 and P8, liquid crystal spatial light modulator 115).
It would have been obvious to PHOSITA before the effective filing date of the claimed invention to modify the combination of Balzarotti, Wang, and Tang with the beam shaping of Tang. PHOSITA would have known about the uses of beam shaping as disclosed by Tang and how to use them to modify the combination of Balzarotti, Wang, and Tang. PHOSITA would have been motivated to do this as a use of known technique to improve similar device in the same way (See MPEP § 2143 (I)(C)), specifically the use of beam shaping for uniform distribution of the light pattern.
Regarding Claim 29, the combination of Balzarotti, Wang, and Tang discloses Claim 27, and Tang further discloses:
…wherein a second phase pattern is generated on the active surface of the beam shaping device (Tang, FIG. 1 and P7, Paragraph 5, “The phase distribution distributed on the liquid crystal spatial light modulator 113 is adjusted by the control module 130 to change the direction, period and initial phase of the light field of the excitation light, so that the uniformly distributed excitation light structure light and light fields and evenly distributed loss light structure light and light The field has the same direction and period, and the initial phase is different by a half period”).
It would have been obvious to PHOSITA before the effective filing date of the claimed invention to modify the combination of Balzarotti, Wang, and Tang with the second pattern of Tang. PHOSITA would have known about the uses of a second pattern as disclosed by Tang and how to use them to modify the combination of Balzarotti, Wang, and Tang. PHOSITA would have been motivated to do this as a use of known technique to improve similar device in the same way (See MPEP § 2143 (I)(C)), specifically the use of other patterns for particular lighting distributions for measurement and analysis.
Regarding Claim 30, the combination of Balzarotti, Wang, and Tang discloses Claim 29, and Tang further discloses:
…wherein the second phase pattern is formed as a ring extending in a circumferential direction to the optical axis (Tang, FIGS. 2(a)-2(c) and FIG. 1 and P6, Paragraph 5, “the light exiting surface P1 of the first flat Gaussian beam shaper 113, the light receiving surface P2 of the liquid crystal spatial light modulator 115, the front focal plane P4 of the objective lens 1123, and the light exit surface of the second flat Gaussian beam shaper 114. P5, the light outgoing plane P6 of the Wollaston prism 116 and the photosensitive surface P7 of the area array detector 123 are on the conjugate plane”).
It would have been obvious to PHOSITA before the effective filing date of the claimed invention to modify the combination of Balzarotti, Wang, and Tang with the second pattern of Tang. PHOSITA would have known about the uses of a second pattern as disclosed by Tang and how to use them to modify the combination of Balzarotti, Wang, and Tang. PHOSITA would have been motivated to do this as a use of known technique to improve similar device in the same way (See MPEP § 2143 (I)(C)), specifically the use of other patterns for particular lighting distributions for measurement and analysis.
Regarding Claim 34, the combination of Balzarotti and Wang discloses Claim 33, but does not explicitly disclose:
…wherein the at least one beam deflection device is configured to reposition the excitation light distribution relative to the deactivation light distribution and to position the local minimum of the excitation light distribution differently more than once within a vicinity of the local intensity minimum of the deactivation light distribution.
However, Tang, in a similar field of endeavor (Uniform Structured Light Illumination Based STED Parallel Micro-imaging System, Has Image Rebuilding Module For Performing Image Reconstruction By Using SIM Frequency Domain Spectrum Map To Realize STED Microscopic Imaging Process), discloses:
…wherein the at least one beam deflection device is configured to reposition the excitation light distribution relative to the deactivation light distribution and to position the local minimum of the excitation light distribution differently more than once within a vicinity of the local intensity minimum of the deactivation light distribution (Tang, FIG. 1 and P8, Paragraph 6, “The Wollaston prism 114 is rotated, and the phase map loaded on the liquid crystal spatial light modulator 115 is rotated accordingly, so that the structured light field fringes of the excitation light and the loss light at the focal plane of the objective lens 1123 can be located in different directions to generate different directions”).
It would have been obvious to PHOSITA before the effective filing date of the claimed invention to modify the combination of Balzarotti and Wang with the stationary deactivation light of Tang. PHOSITA would have known about the uses of stationary deactivation light as disclosed by Tang and how to use them to modify the combination of Balzarotti and Wang. PHOSITA would have been motivated to do this as a use of known technique to improve similar device in the same way (See MPEP § 2143 (I)(C)), specifically the use a stationary deactivation light to generate different directions and angles of light in conjunction with the activation light.
Regarding Claim 35, the combination of Balzarotti and Wang discloses Claim 33, and Balzarotti further discloses:
…wherein the device comprises a first beam shaping device for generating the excitation light distribution (Balzarotti, FIG. 2, Description of FIG. 2, “shaped by a vortex-phase mask”) and …
The combination of Balzarotti and Wang discloses the above but does not explicitly disclose:
… a second beam shaping device for generating the deactivation light distribution independently of the generation of the excitation light distribution.
However, Tang, in a similar field of endeavor (Uniform Structured Light Illumination Based STED Parallel Micro-imaging System, Has Image Rebuilding Module For Performing Image Reconstruction By Using SIM Frequency Domain Spectrum Map To Realize STED Microscopic Imaging Process), discloses:
a second beam shaping device for generating the deactivation light distribution independently of the generation of the excitation light distribution (Tang, FIG. 1 and P5, Paragraph 5, Gaussian beam shaper 114).
It would have been obvious to PHOSITA before the effective filing date of the claimed invention to modify the combination of Balzarotti and Wang with the second beam shaping device of Tang. PHOSITA would have known about the uses of beam shaping devices as disclosed by Tang and how to use them to modify the combination of Balzarotti and Wang. PHOSITA would have been motivated to do this as a use of known technique to improve similar device in the same way (See MPEP § 2143 (I)(C)), specifically the use a second beam shaping device to control and direct light.
Claim 21 is rejected under 35 U.S.C. 103 as being unpatentable over Balzarotti (Francisco Balzarotti et al. ,Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes.Science355,606-612(2017).DOI:10.1126/science.aak9913), in view of Wang (“Stimulated emission depletion microscopy with array detection and photon reassignment,” Wensheng Wang et. al., Optics and Lasers in Engineering, V. 129, June 2020, https://www.sciencedirect.com/science/article/pii/S014381661931797X), and in further view of Hell (US20140042340).
Regarding Claim 21, the combination of Balzarotti and Wang discloses Claim 16, but does not explicitly disclose:
…wherein a region between two local maxima of the deactivation light distribution adjacent to the local intensity minimum of the deactivation light distribution (Hell, FIGS. 9-10 and [0093], minimum 19) is extended further, particularly at least 10% further, at least in one spatial direction than a corresponding region between two local maxima of the excitation light distribution adjacent to the local minimum of the excitation light distribution Balzarotti discusses it a bit throughout its system of finding low spots, but not to the degree the Examiner would prefer.
However, Hell, in a similar field of endeavor (tracking a movement of a particle in a sample), discloses:
…wherein a region between two local maxima of the deactivation light distribution adjacent to the local intensity minimum of the deactivation light distribution (Hell, FIGS. 9-10 and [0093], minimum 19) is extended further, particularly at least 10% further, at least in one spatial direction than a corresponding region between two local maxima of the excitation light distribution adjacent to the local minimum of the excitation light distribution (Hell, FIGS. 9-10 and [0093], “The distance of the maxima of the light intensity distribution 18 in x-direction, however, is somewhat smaller than a distance between the maxima of the intensity distribution 35 of the STED light 36 in x-direction. This corresponds to a longer wavelength of the STED light 36 as compared to the light 4. Further, the intensity distribution 35 exceeds a saturation intensity I.sub.S outside a region whose diameter is indicated by a double-headed arrow 37 in FIG. 9. Thus, only if located within this region, a particle is effectively driven to emit photons by the light 4, as the emission of photons which are detected is inhibited by the STED light 36 everywhere else”
It would have been obvious to PHOSITA before the effective filing date of the claimed invention to modify the combination of Balzarotti and Wang with light distribution control of Hell. PHOSITA would have known about the uses of light distribution control as disclosed by Hell and how to use them to modify the combination of Balzarotti and Wang. PHOSITA would have been motivated to do this as a use of known technique to improve similar device in the same way (See MPEP § 2143 (I)(C)), specifically the use of modifying the intensity distributions to better track particles.
Claim 25 is rejected under 35 U.S.C. 103 as being unpatentable over Balzarotti (Francisco Balzarotti et al. ,Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes.Science355,606-612(2017).DOI:10.1126/science.aak9913), in view of Wang (“Stimulated emission depletion microscopy with array detection and photon reassignment,” Wensheng Wang et. al., Optics and Lasers in Engineering, V. 129, June 2020, https://www.sciencedirect.com/science/article/pii/S014381661931797X), and in further view of Prasad (US20160291343).
Regarding Claim 25, the combination of Balzarotti and Wang discloses Claim 16, and Balzarotti further discloses:
… wherein the 2D donut is generated by phase modulation of the deactivation light with a first phase pattern (Balzarotti, FIG. 2(b), Description of FIG.2, “Intensity modulation and deflection, as well as the photon counting, are controlled by a field-programmable gate array (FPGA)”) …
The combination of Balzarotti and Wang discloses the above but does not explicitly disclose:
…the first phase pattern comprising a phase increasing in a circumferential direction with respect to an optical axis, particularly continuously, from 0 to 2πn, wherein n is a natural number greater than 1.
However, Prasad, in a similar field of endeavor (biological microscopy), discloses:
…the first phase pattern comprising a phase increasing in a circumferential direction with respect to an optical axis, particularly continuously, from 0 to 2πn, wherein n is a natural number greater than 1 (Prasad, FIGS. 4A-4N and [0093], “While the PSF remains nearly shape and size invariant as it rotates at the rate of 1/L radians per unit defocus phase completing a full rotation for a defocus change of about 2πL radians, in consistency with the approximate result (6), there is clear evidence of differential rotation and slow spreading”).
It would have been obvious to PHOSITA before the effective filing date of the claimed invention to modify the combination of Balzarotti and Wang with the rotation technique of Prasad. PHOSITA would have known about the uses of rotation techniques as disclosed by Prasad and how to use them to modify the combination of Balzarotti and Wang. PHOSITA would have been motivated to do this as a use of known technique to improve similar device in the same way (See MPEP § 2143 (I)(C)), specifically the use rotation to account for the defocus.
Claim 28 is rejected under 35 U.S.C. 103 as being unpatentable over Balzarotti (Francisco Balzarotti et al. ,Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes.Science355,606-612(2017).DOI:10.1126/science.aak9913), in view of Wang (“Stimulated emission depletion microscopy with array detection and photon reassignment,” Wensheng Wang et. al., Optics and Lasers in Engineering, V. 129, June 2020, https://www.sciencedirect.com/science/article/pii/S014381661931797X), in further view of Tang (CN108181235A), and in further view of Gould (Gould, “Adaptive optics enables 3D STED microscopy in aberrating specimens,” Travis J. Gould et al., Opt Express. 2012 Sep 10, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3635694/).
Regarding Claim 28, the combination of Balzarotti, Wang, and Tang discloses Claim 27 but does not explicitly disclose:
…wherein an orientation of a blazed grating in an outer region of the active surface of the beam shaping device is adjusted.
However, Gould, in a similar field of endeavor (3D STED microscopy), discloses:
…wherein an orientation of a blazed grating in an outer region of the active surface of the beam shaping device is adjusted (Gould, FIG. 1 and P21001, Description of FIG. 1, “a circular blazed grating (for off-axis phase modulation) that defines the active area,” and P21002, Paragraph 2, “In our setup an active pupil was defined on the SLM by creating such an off-axis hologram (Fig. 1 insets) – a blazed grating of circular outline that diffracts light off the principal reflection axis into the first diffraction order – and the diffracted light was focused into the sample”).
It would have been obvious to PHOSITA before the effective filing date of the claimed invention to modify the combination of Balzarotti, Wang, and Tang with the blazed grating of Gould. PHOSITA would have known about the uses of blazed grating as disclosed by Gould and how to use them to modify the combination of Balzarotti, Wang, and Tang. PHOSITA would have been motivated to do this as a use of known technique to improve similar device in the same way (See MPEP § 2143 (I)(C)), specifically the use of a blazed grating for focusing with a STED apparatus.
Claim 31 is rejected under 35 U.S.C. 103 as being unpatentable over Balzarotti (Francisco Balzarotti et al. ,Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes.Science355,606-612(2017).DOI:10.1126/science.aak9913), in view of Wang (“Stimulated emission depletion microscopy with array detection and photon reassignment,” Wensheng Wang et. al., Optics and Lasers in Engineering, V. 129, June 2020, https://www.sciencedirect.com/science/article/pii/S014381661931797X), in further view of Tang (CN108181235A), and in further view of Betzig (WO2016054118A1).
Regarding Claim 31, the combination of Balzarotti, Wang, and Tang discloses Claim 30 but does not explicitly disclose:
…wherein the ring comprises a plurality of segments, wherein adjacent segments respectively comprise a phase difference of π to each other.
However, Betzig, in a similar field of endeavor (structured plane illumination microscopy), discloses:
…wherein the ring comprises a plurality of segments, wherein adjacent segments respectively comprise a phase difference of π to each other (Betzig, FIGS. 45A-45D and [00284], “FIGs. 45A, 45B, 45C, and 45D, which are analogous to FIG. 17E, show observable regions of reciprocal space representations of specimen structure shifted from the reciprocal space origin,” FIGS. 52A-52C and [00310], “FIGs. 52B and 52C shows spatially-structured excitation patterns with a saturation factors of 3.0 and 0.5, respectively, that can be applied to the hypothetical sample in two successive read-out steps, where the spatially-structured excitation pattern of FIG. 52B is out-of-phase by π with the spatially-structured activation pattern and where the spatially-structured excitation pattern of FIG. 52C is out-of- phase by π with the spatially-structured excitation pattern of FIG. 52B”).
It would have been obvious to PHOSITA before the effective filing date of the claimed invention to modify the combination of Balzarotti, Wang, and Tang with the phase pattern of Betzig. PHOSITA would have known about the uses of phase patterns as disclosed by Betzig and how to use them to modify the combination of Balzarotti, Wang, and Tang. PHOSITA would have been motivated to do this as a use of known technique to improve similar device in the same way (See MPEP § 2143 (I)(C)), specifically to extract the information available in the fluorescence light gathered in each phase and then used to construct image of the sample comprised of those phases.
Claim 32 is rejected under 35 U.S.C. 103 as being unpatentable over Balzarotti (Francisco Balzarotti et al. ,Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes.Science355,606-612(2017).DOI:10.1126/science.aak9913), in view of Wang (“Stimulated emission depletion microscopy with