DETAILED ACTION
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 02/10/2026 has been entered.
Status of claim rejections
The rejections of the claims under 35 USC 103 have been maintained in view of Applicant’s arguments and amendments in the response filed 02/10/2026.
Claim Interpretation
The examiner is interpreting the claims as set forth below:
Claim 1 has been amended to recite, inter alia, “wherein said tissue sample has not been hydrogel embedded and/or has not been fixed with glutaraldehyde”. The examiner is interpreting this new limitation as intended use of the claimed solution, as the instant claims are drawn to a product (i.e., a solution) and not a method (such as using the solution to prepare a tissue sample that has not been hydrogel embedded and/or not fixed with glutaraldehyde). The invention does not actually require a tissue sample, only the buffer solution with a pH below 8 and a detergent and this interpretation is reflected in the rejections/response to arguments below.
Maintained 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:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
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 as of the effective filing date of the claimed invention(s) 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 as of the effective filing date of the later invention 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.
Claims 12, and 14-15 are rejected under 35 U.S.C. 103 as being unpatentable over Gradinaru et al (US10794802B2; see also US 20170199104) in view of Calbiochem® ("Detergents: a guide to the properties and uses of detergents in biology and biochemistry"; May 2007; prior art of record; retrieved online from https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/412/140/cb0068-ms.pdf).
Gradinaru teaches enhanced tissue clearing for improved visualization of various constituents of the tissue (e.g., cells, nucleic acids, and other small molecules) (see abstract; see paragraph 0036). Gradinaru teaches various incubation solutions for clearing including: a borate-buffered solution having 4-10% SDS and 0.2 M boric acid buffer, or boric acid wash buffer (BBT), where the BBT includes 0.2 M boric acid buffer and 0.1-0.25% Triton X-100 (vol/vol) (a solution comprising a boric acid buffer solution and a detergent as in claim 1; see paragraph 0036; 0082; 0164; 0366). Gradinaru teaches that the pH of the BBT buffer is 6.5-9.5 (see paragraph 0036). Please note a pH below 8 as instantly claimed is encompassed by the disclosure of Gradinaru. In the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists. In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990) (see MPEP 2144.05(I)).
Gradinaru does not explicitly teach that the detergent is any one of n-Octyl-N,N-dimethvl-3-ammonio-1-propanesulfonate, n-Decyl-N,N-dimethvl-3-ammonio-1-propanesulfonate, n-Dodecyl-N, N- dimethyl-3-ammonio-1-propanesulfonate, n-Tetradecyl-N, N-dimethvl-3-ammonio-1-propanesulfonate, and n-Hexadecyl-N,N-dimethvl-3-ammonio-1-propanesulfonate.
However, Calbiochem® teaches various detergents for biochemical and biological uses (title). Calbiochem® teaches that Zwitterionic detergents are unique in that they offer the combined properties of ionic and non-ionic detergents (pg. 10, paragraph 1). Like non-ionic detergents the zwittergents, including CHAPS and the ZWITTERGENT® Detergent 3-X series, do not possess a net charge, they lack conductivity and electrophoretic mobility, and do not bind to ion-exchange resins (pg. 10, paragraph 1). However, like ionic detergents, they are efficient at breaking protein-protein interactions (pg. 10, paragraph 1). Calbiochem® further teaches the ZWITTERGENT® Detergent 3-X detergents include Zwittergent® 3-10 (also known as n-Decyl-N, N-dimethvl-3-ammonio-1-propanesulfonate; see also Applicant’s specification pg. 20; as in claim 12) (see pg. 28 and 42).
Therefore, it would have been prima facie obvious to one of ordinary skill at the time of filing to modify the 3D imaging preparation solution as taught by Gradinaru with the n-Decyl-N, N-dimethvl-3-ammonio-1-propanesulfonate of Calbiochem® to arrive at the claimed invention. As the claimed invention relies on the creation of a 3D imaging preparation solution using a detergent, one of ordinary skill would have been motivated to perform a simple substitution of one known element (the detergent of Gradinaru) with another (the n-Decyl-N, N-dimethvl-3-ammonio-1-propanesulfonate of Calbiochem®) with a reasonable expectation of success. One of ordinary skill would have been motivated to make the modification because Calbiochem® teaches that n-Decyl-N, N-dimethvl-3-ammonio-1-propanesulfonate advantageously do not possess a net charge, lack conductivity electrophoretic mobility, do not bind to ion-exchange resins and are efficient at breaking protein-protein interactions.
Neither reference teach that the tissue sample has not been hydrogel embedded and/or not fixed with glutaraldehyde. However, the instant claims are drawn to a product (i.e., a solution) and not a method (such as using the solution to prepare a tissue sample that has not been hydrogel embedded and/or not fixed with glutaraldehyde). The claimed solution does not actually require a tissue sample, only the buffer solution with a pH below 8 and a detergent which is taught by Gradinaru and Calbiochem.
Regarding claim 14, Gradinaru teaches the buffered solution has a pH of 6.5-9.5 (see paragraph 0036) and Xu teaches use of pH at 7.5. The pH as claimed (of about 6 to a pH below 8) is encompassed by the disclosure of Gradinaru and Xu. In the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists. In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990) (see MPEP 2144.05(I)). Furthermore, generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (see MPEP 2144.05(II)).
Regarding claim 15, Calbiochem® teaches Zwittergent® 3-10, which is n-Decyl-N, N-dimethvl-3-ammonio-1-propanesulfonate (see also Applicant’s specification pg. 20).
Accordingly, the claimed invention was prima facie obvious to one of ordinary skill at the time of filing, especially in the absence of evidence to the contrary.
Response to Arguments
Applicant's arguments filed 02/10/2026 have been fully considered but they are not persuasive.
On pg. 5-7 of the remarks, Applicant argues the claims are not obvious over Gradinaru and Calbiochem. Specifically, Applicant argues that the claimed invention exhibits the unexpected property of enabling preparation of tissues for 3D imaging without need of additional fixation by hydrogel or glutaraldehyde. Applicant cites to the specification at pg. 2 to show disadvantages of fixing of tissue including decreased sample quality and from this Applicant argues neither reference contemplates the unexpected property and that Gradinaru teaches importance of fixing or embedding the sample prior to clearing. Applicant also argues that a PHOSITA would not substitute the detergent of Gradinaru with zwitterionic detergent as claimed with a reasonable expectation of success.
In response, the examiner disagrees. First, as interpreted above, the instant claims are drawn to a product (i.e., a solution) and not a method (such as using the solution to prepare a tissue sample that has not been hydrogel embedded and/or not fixed with glutaraldehyde). Put simply, the claimed invention does not actually require a tissue sample (such that the lack of glutaraldehyde or hydrogel embedding does not impart any structural , only the boric acid buffer solution with a pH below 8 and a detergent, which is taught by the combination of references cited in the rejection above.
Even if, arguendo, the claimed solution were to require a tissue sample, Xu et al (Scientific Reports, 2017, 7:9895; prior art of record) evidences that hydrogel-based tissue clearing methods like PACT (i.e., the protocol used by Gradinaru) and CLARITY provide conditions for antibody labelling of tissue markers but for long-term imaging, the deformation of tissues cause by hydrogel expansion during clearing limits usefulness for evaluating fine structures such as microglial branches and neuronal processes (see pg. 2, paragraph 2). Xu evidences that avoiding the use of hydrogel increases speed of clearing compared to traditional methods and that removing hydrogel perfusion and embedding steps improves the signal to noise ratio, depth of tissue penetration, speed of processing, long-term retention of fluorescent signal, and preservation of cytoarchitecture (see pg. 2, paragraph 3). Thus, the prior art recognizes avoiding use of hydrogel embedding, specifically for the reasons and advantages that Applicant cites are “unexpected” (i.e., avoiding deformation of the tissue, increases speed of clearing compared to traditional methods and that removing hydrogel perfusion and embedding steps improves the signal to noise ratio, depth of tissue penetration, speed of processing, long-term retention of fluorescent signal, and preservation of cytoarchitecture).
Furthermore, Gradinaru teaches enhanced tissue clearing for imaging using borate buffered solutions with a detergent at pHs below 8, and Calbiochem specifically teaches that zwitterionic detergents, like non-ionic detergents (e.g., SDS), do not possess a net charge, they lack conductivity and electrophoretic mobility, and do not bind to ion-exchange resins (pg. 10, paragraph 1). However, like ionic detergents, they are efficient at breaking protein-protein interactions. As such, one of ordinary skill would have been motivated to use a zwittergent detergent as instantly claimed because the detergent has advantageous properties attributed to both non-ionic detergents and ionic detergents alike. As such, the rejections are maintained as set forth above.
Conclusion
NO CLAIMS ALLOWED.
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/G.C.R./Examiner, Art Unit 1651
/THOMAS J. VISONE/Supervisory Patent Examiner, Art Unit 1672
