DETAILED ACTION
Notice of Pre-AIA or AIA Status
1. The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Request for Continued Examination
2. 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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 12/17/25 has been entered.
Information Disclosure Statement
3. The information disclosure statement (IDS) submitted on 12/17/25 was filed and entered. The submission is in compliance with the provisions of 37 CFR 1.97 and has been considered by the Examiner.
Claim Status
4. The amendment, filed 12/17/25, has been entered. Claims 1-25 are pending and under examination. Claims 1 and 7 are amended.
Withdrawal of Objections/Rejections
5. The following are withdrawn from the Office Action, filed 09/17/25:
The rejection of claim 7 under 35 U.S.C. 112(b), as being indefinite, found on page 15 at paragraph 15, is withdrawn in light of Applicant’s amendments thereto.
New Rejection: Claim Rejections - 35 USC § 112
6. 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.
7. Claims 1-25 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.
Newly amended claim 1 recites “…(b) bulk labeling amino acids of non-identical molecules in the expanded sample with at least one reagent to introduce contrast”; which is indefinite because it is unclear how the bulk label would label the amino acids of as little as one of each type of particular type of protein (e.g. one particular cell membrane protein and one cytoplasmic enzyme would constitute labeling non-identical molecules), but not all of said cell membrane proteins because those would be identical molecules (e.g. one cytochrome C but not all the cytochrome C and not cytochrome C on any more than one cell because they would not be non-identical). Further, it is unclear if the limitation encompasses labeling all the amino acids of at least two non-identical molecules or labeling all of one particular amino acid (e.g. alanine) which would be expected to differ in location among different types of proteins, but how the label would only label only one of them is unclear because otherwise the bulk label is labeling identical molecules. Dependent claims do not clarify the issue, thus clarification is required to remove scope ambiguity and ascertain the metes and bounds.
Maintained Claim Rejections - 35 USC § 102
8. 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.
9. The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
10. Claims 1-12, 14-19, and 21-22 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Boyden et al. 2018 (WO 2018/157074).
Boyden teaches methods for preparing an expanded biological specimen, including cells and/or tissues, suitable for microscopic analysis, by physically expanding the sample and bulk labeling subcellular features, including proteins of interest (i.e. amino acids; e.g. see page 1, line 24 to page 2, line 5; page 7, lines 5-15; page 10, lines 18-21; page 12, lines 28-31; page 14, line 28 to page 15, line 1; meeting limitations found in instant claims 1, 2, and 8). Boyden teaches the use of fluorescent dyes for non-specific, bulk labeling of proteins, nucleic acids and/or carbohydrates (i.e. also non-identical molecules; e.g. page 7, lines 16-23; page 11, lines 17-18; page 14, line 28 to page 15, line 5; and page 15, lines 30-33; meeting limitations found in newly amended claim 1, along with dependent claim 8). Boyden also teaches samples stained with hematoxylin and eosin (i.e. H&E stains; both of which bulk label non-identical molecules, also meeting limitation in newly amended claim 1; see page 7, lines 16-23). Boyden teaches exemplary biological tissues include brain tissue and includes samples derived from humans, animals, and/or plants (e.g. page 11, line 30 to page 12, line 5; meeting limitations found in claims 15 and 16). Boyden teaches the use of any imaging device and a computer to obtain images, analyze and compare results from the images, for example from observed nuclei (i.e. subcellular component, specifically) of the expanded biological samples (e.g. see page 2, lines 3-10; page 6, lines 27-30; page 14, lines 32-33; and page 23, lines 1-7; meeting limitations of instant claims 4, 12 and 14). Boyden teach expansion microscopy is used to increase the resolution of conventional optical microscopy by 4-5 fold (e.g. page 7, lines 5-13; and page 35, lines 12-14; meeting limitations found in instant claims 1 and 4). Boyden teaches the methods are used with formalin fixed paraffin embedded samples (i.e. chemical fixation; e.g. page 7, lines 16-23; and page 17, lines 11-12; meeting limitations found in instant claim 3). Boyden teaches the sample in contacted with one or more detectable labeling reagents including fluorophores and fluorescent dyes, with or without antibodies (i.e. encompasses, but is not limited to immunofluorescence; e.g. see page 15, lines 7-17; and page 16, lines 1-14; meeting limitations found in instant claims 1, 4, 5, and 11). Boyden teaches the use of NHS esters and acrylate which allows primary amines to covalently bind the carboxylic groups and form charge-neutral amides in a swellable gel (e.g. page 18, lines 3-13; and page 27, lines 15-20; meeting limitations found in instant claims 9 and 10). Boyden teaches proteins in the sample of interest can be modified with protein-reactive groups comprising a click group and gel-reactive groups comprising a complementary click group, in separate steps using click chemistry (e.g. page 15, lines 24-26; page 17; lines 26-31; and page 18, lines 19-25; meeting limitations found in instant claims 6 and 7). Boyden teaches the use of swellable and non-swellable polymers, with and without crosslinking, and with or without anchoring (i.e. Boyden does not require crosslinking or anchoring; e.g. page 7, lines 5-15; page 26, line 31 to page 27, line 10; and Boyden independent claim 1; meeting limitations found in instant claims 17, 18, 21, and 22). Boyden teach the use of acrylamides and polyacrylamides crosslinkers and provides an example using concentrations of 0.1% (w/w); (e.g. page 19, lines 1-5 and lines 12-16; page 27, lines 8-10; and page 32; meeting limitations found in instant claim 19). In addition, Boyden teaches that embedding a sample in a non-swellable polymer comprises permeating one or more monomers or other precursors throughout the sample and polymerizing and/or crosslinking the monomers or precursors to form the non-swellable polymer and that embedding the expanded sample in a non-swellable polymer prevents conformational changes during sequencing despite salt concentration variation (page 27, lines 1-10). Boyden also teaches fixation of the biological specimen in the presence of hydrogel subunits crosslinks the biomolecule of the specimen to the hydrogel subunits, and thereby secures molecular components in place and preserves the tissue architecture and cell morphology of the sample (e.g. page 19, lines 17-20). Boyden teaches the proteins of interest (i.e. amino acids) may be labeled post-expansion if nonspecific proteolysis of the expansion microscopy is replaced with modified post-gelation homogenization treatments (e.g. see page 20, lines 12-15; meeting limitations in new amended claim 1).
Therefore, Boyden anticipates the invention as claimed.
Applicant’s Arguments and Response to Arguments
11. All of Applicant’s arguments have been considered but were not deemed persuasive; accordingly, the rejection is maintained for reasons of record. For example:
With regards to the arguments that Boyden is silent with regards to bulk labeling of non-identical molecules including the sections of Boyden reproduced by Applicant (see Remarks, page 5-6); the Office disagrees with Applicant’s limited interpretation of Boyden and notes (1) it is not clear what this limitation includes or excludes (see 112(b) rejection above); (2) claim 1 does not require any particular labelling agent; and (3) Boyden teaches the general use of fluorescent dyes to detect biomolecules, including proteins, which would be recognized as encompassing the general staining (i.e. non-specific, global labeling) of those proteins, as further evidenced by the state of the art (see below). Therefore, these arguments are not persuasive because Boyden is not limited to antibody-directed immunofluorescence, immunohistochemistry and immunocytochemistry and/or RNA/DNA hybridization, as asserted. In addition, claim 9 requires the reagent to be amine-reactive, thiol-reactive, carboxyl-reactive, tyrosine-reactive and/or glutamine-reactive each of which would considered by the skilled artisan to function as either as “bulk” (i.e. all amino acids in different proteins) or “specific” (i.e. only tyrosine but in different proteins) and varying levels in between. Therefore, Applicant’s assertion that the definition in the instant specification negates the interpretation of labeling in the prior art is still unpersuasive.
Moreover, contrary to Applicant’s assertion, Boyden is not restricted to immunostaining (i.e. use of specific antibodies) just because they describe one or more embodiments using a highly specific immunostaining agent (e.g. see Boyden, page 16, line 3-6, which explicitly states Boyden is not limited to such techniques). See MPEP 2131 which states that disclosed examples and preferred embodiments do not constitute a teaching away from a broader disclosure or nonpreferred embodiments; see In re Susi, 440 F.2d 442, 169 USPQ 423 (CCPA 1971); and In re Gurley, 27 F.3d 551, 554, 31 USPQ2d 1130, 1132 (Fed. Cir. 1994). Further, “[t]he prior art’s mere disclosure of more than one alternative does not constitute a teaching away from any of these alternatives because such disclosure does not criticize, discredit, or otherwise discourage the solution claimed….” In re Fulton, 391 F.3d 1195, 1201, 73 USPQ2d 1141, 1146 (Fed. Cir. 2004).
Furthermore, Applicant’s position in not supported by the state of the art, as evidenced by for example, Miller et al. 2006 (Protein stains for proteomic applications: Which, when, why? Proteomics 6:5385-5408) published over a decade before the effective filing date and which reviews the sensitivity and dynamic range for the most commonly used fluorescent dyes for general protein staining (e.g. see abstract; Figure 1; and section 2). Miller teaches these dyes are the main alternative and complement to immunological procedures for detecting proteins (i.e. fluorescent dyes are used with and/or without antibodies; e.g. page 5385, left column). Miller teaches well-known, common examples include cyanine-based fluorescent dyes which covalently label lysine residues via an amide linkage (e.g. section 2.1.4.1); TMB (i.e. monobromobimane) and FlaSH® dyes that fluorescently label cysteine residues via a reaction with its thiol or maleimide groups (e.g. section 2.1.4.2); Direct Blue 71 which fluorescently stains proteins with a sensitivity of 5-20 nanograms while SYPRO Ruby has a detection sensitivity of less than one nanogram per square millimeter (e.g. section 2.2.2); and proprietary fluorescent stains such as Pro-Q Diamond® which affords wider specificity, higher sensitivity and an easy protocol to detect proteins without context specificity (e.g. section 3.1). More recently, Kozma et al. 2019 (Fluorogenic probes for super-resolution microscopy; Organic and Biomolecular Chemistry; 17:215) reviewed the use of fluorogenic probes (i.e. fluorophores) for super-resolution microscopy (see abstract) and teaches synthetic fluorophores can be applied to virtually any biomolecular structure in contrast to other tags that are often limited to terminal labeling of proteins (e.g. see Introduction, page 215). Kozma teach another advantage of the use of fluorophores is that they (e.g. FlaSH® dyes) preserve the biological function of a protein that would otherwise be disrupted by using antibody binding (e.g. page 220, right column). Accordingly, the state of the art (i.e. what one skilled in the art would reasonably know) supports that the ordinary artisan would recognize (a) fluorescent dyes are routinely used with and without antibodies and (b) there are advantages to using fluorescent dyes without antibodies (e.g. preserves biological function of the protein).
Accordingly, it remains the Office’s position that the ordinary artisan, reading Boyden (in its entirety, but focusing on the excerpts below), would easily recognize that the fluorescent dyes taught by Boyden would not be limited to immunofluorescent dyes (i.e. attached to antibodies) as asserted by Applicant, but rather would easily and immediately envision that the fluorescent dyes taught by Boyden encompassed commonly used fluorescent dyes for the general staining of proteins.
see Boyden at:
Page 11
PNG
media_image1.png
112
670
media_image1.png
Greyscale
Page 14
PNG
media_image2.png
176
692
media_image2.png
Greyscale
Page 15
PNG
media_image3.png
175
705
media_image3.png
Greyscale
PNG
media_image4.png
91
711
media_image4.png
Greyscale
Page 16:
PNG
media_image5.png
315
680
media_image5.png
Greyscale
Accordingly, Boyden teaches macromolecules, including fluorescent dyes (i.e. fluorophores), may be provided that promote the visualization of cellular biomolecules, including proteins, and thereby meets the broadest reasonable interpretation of the newly added limitation of claim 1 because the general stains would detect all proteins, at least some of which are “non-identical” to each other. Therefore, these arguments are not persuasive and it remains the Office’s position that Boyden clearly envisioned both non-specific (i.e. bulk) and specific (i.e. see claims 9 and 14) labeling of subcellular components, including proteins, in their methods for expansion microscopy, as set forth above.
Therefore, all of Applicant’s arguments have been considered but were not deemed persuasive; accordingly, the rejection is maintained for reasons of record.
Maintained Rejection: Claim Rejections - 35 USC § 103
12. 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.
13. 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.
14. Claims 1-24 are rejected under 35 U.S.C. 103 as being unpatentable over Boyden et al. 2018 (WO 2018/157074) in view of Wiersma et al. 1994 (High strength poly(meth)acrylamide copolymer hydrogels; Polymer bulletin 33: 615-622).
The teachings of Boyden are outlined above.
With regards to the expanded factor of 12-24 in each direction, found in dependent claim 13; Boyden teaches expansion microscopy is routinely used to increase the resolution of conventional optical microscopy by at least 4-5 fold and recognizes the additional advantages of expansion microscopy such as tissue clearing, resolution improvement, and higher tolerance to sectioning error (i.e. the amount of expansion is a results-effective variable; see page 7, lines 5-13; and page 35, lines 12-14). Therefore, it is the Office’s position that this limitation amounts to routine optimization of a known, results-effective variable (i.e. the amount of expansion in expansion microscopy) via routine experimentation which would be recognized as part of the ordinary capabilities of one skilled in the art and it is well established that it is not inventive to discover the optimum or workable ranges by routine experimentation; see MPEP § 2144.05(II)(B) and KSR International Co. v. Teleflex Inc., 127 S. Ct. 1727, 1741 (2007).
With regards to the concentrations found in dependent claims 23 and 24; MPEP 2144.05 states, 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) (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature of 100°C and an acid concentration of 10%.); see also Peterson, 315 F.3d at 1330, 65 USPQ2d at 1382 ("The normal desire of scientists or artisans to improve upon what is already generally known provides the motivation to determine where in a disclosed set of percentage ranges is the optimum combination of percentages."); In re Hoeschele, 406 F.2d 1403, 160 USPQ 809 (CCPA 1969) (Claimed elastomeric polyurethanes which fell within the broad scope of the references were held to be unpatentable thereover because, among other reasons, there was no evidence of the criticality of the claimed ranges of molecular weight or molar proportions.). For more cases applying this principle, see Merck & Co. Inc. v. Biocraft Laboratories Inc., 874 F.2d 804, 10 USPQ2d 1843 (Fed. Cir.), cert. denied, 493 U.S. 975 (1989); In re Kulling, 897 F.2d 1147, 14 USPQ2d 1056 (Fed. Cir. 1990); and In re Geisler, 116 F.3d 1465, 43 USPQ2d 1362 (Fed. Cir. 1997)."
Therefore, the difference between the prior art and the invention encompasses wherein the polymer crosslinkers in the hydrogel are piperazine diacrylamide (e.g. see dependent claim 20).
However, Wiersma teaches cross-linking a hydrogel with piperazine diacrylamide yields a strong, glassy hydrogel useful in optical elements (e.g. see page 615, abstract and page 621, conclusions). Wiersma teaches the importance of producing hydrogels with high optical refractive indices and good clarity (e.g. page 615, introduction). Wiersma teaches increasing cross-linker concentration gives a stronger network, whereas using a lower initiator concentration increases phase separation and inhomogeneity (i.e. concentration of cross-linker is a results-effective variable; e.g. page 615, introduction).
Therefore, it would have been prima facie obvious, before the effective filing date of the claimed invention, to a person of ordinary skill in the art, to modify the methods for preparing a biological sample for expansion microscopy, as taught by Boyden, by cross-linking the hydrogel with piperazine diacrylamide, thereby arriving at the claimed invention, in order to produce a strong, glassy hydrogel with good clarity and a high optical refractive index, as taught by Wiersma. Therefore, each and every element is taught in the prior art and the combination has a beneficial result. However, the combination amounts to no more than a predictable use of prior art elements according to their established functions. The person of ordinary skill in the art would have been motivated to make the modification, with at least a reasonable expectation of success, because cross-linking hydrogels with piperazine diacrylamide was already recognized as producing a strong glassy hydrogel with improved properties and useful in optical elements, as taught by Wiersma. Therefore, the combination leads to expected results because each element performs the same function as it does individually.
Additionally, KSR International Co. v. Teleflex Inc., 127 S. Ct. 1727, 1741 (2007), discloses that combining prior art elements according to known methods to yield predictable results, is obvious unless its application is beyond that person's skill. KSR International Co. v. Teleflex Inc., 127 S. Ct. 1727, 1741 (2007) also discloses that the combination of familiar elements according to known methods is likely to be obvious when it does no more than yield predictable results. In the instant case, all elements (i.e. methods for preparing biological samples for expansion microscopy using cross-linked hydrogels and hydrogels cross-linked with piperazine diacrylamide used in optical applications) were known in the art. In addition, combining these elements yields a method wherein each element merely performs the same function as it does separately; thus the results of the combination would be recognized as predictable to one of ordinary skill in the art. Therefore, it would have been obvious to a person of ordinary skill in the art to combine these prior art elements according to a known method to yield predictable results.
Therefore, the claimed invention is prima facie obvious in view of the teachings of the prior art, absent any convincing evidence to the contrary.
Applicant’s Arguments and Response to Arguments
15. All of Applicant’s arguments have been considered but were not deemed persuasive; accordingly, the rejection is maintained for reasons of record. For example:
With regards to the argument that the Office failed to establish a prima facie case of obviousness because the combination of references fails to teach bulk labeling of non-identical molecules (i.e. Wiersma does not cure the deficiencies of Boyden; see Remarks, pages 7-9), the Office disagrees (see above) and reminds Applicant the combination was based on the limitation regarding the use of piperazine diacrylamide, which the combination teaches.
With regards to the observed improved resolution over the electron microscopy photograph as taught by Peddie et al. 2014 (see Remarks and Figure on page 8), the Office notes Boyden in not limited to electron microscopy and Peddie is not part of the rejection. Therefore this argument is not germane.
Therefore, all of Applicant’s arguments have been considered but were not deemed persuasive; accordingly, the rejection is maintained for reasons of record.
Maintained Rejection: Claim Rejections - 35 USC § 103
16. Claims 1-19 and 21-25 are rejected under 35 U.S.C. 103 as being unpatentable over Boyden et al. 2018 (WO 2018/157074) in view of Buffa et al. 2015 (a-b, unsaturated aldehyde of hyaluronan-synthesis, analysis and applications; Carbohydrate Polymers 134:293-299).
The teachings of Boyden are outlined above.
With regards to the expanded factor of 12-24 in each direction, found in dependent claim 13; Boyden teaches expansion microscopy is routinely used to increase the resolution of conventional optical microscopy by at least 4-5 fold and recognizes the additional advantages of expansion microscopy such as tissue clearing, resolution improvement, and higher tolerance to sectioning error (i.e. the amount of expansion is a results-effective variable; see page 7, lines 5-13; and page 35, lines 12-14). Therefore, it is the Office’s position that this limitation amounts to routine optimization of a known, results-effective variable (i.e. the amount of expansion in expansion microscopy) via routine experimentation which would be recognized as part of the ordinary capabilities of one skilled in the art and it is well established that it is not inventive to discover the optimum or workable ranges by routine experimentation; see MPEP § 2144.05(II)(B) and KSR International Co. v. Teleflex Inc., 127 S. Ct. 1727, 1741 (2007).
With regards to the concentrations found in dependent claims 23 and 24; MPEP 2144.05 states, 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) (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature of 100°C and an acid concentration of 10%.); see also Peterson, 315 F.3d at 1330, 65 USPQ2d at 1382 ("The normal desire of scientists or artisans to improve upon what is already generally known provides the motivation to determine where in a disclosed set of percentage ranges is the optimum combination of percentages."); In re Hoeschele, 406 F.2d 1403, 160 USPQ 809 (CCPA 1969) (Claimed elastomeric polyurethanes which fell within the broad scope of the references were held to be unpatentable thereover because, among other reasons, there was no evidence of the criticality of the claimed ranges of molecular weight or molar proportions.). For more cases applying this principle, see Merck & Co. Inc. v. Biocraft Laboratories Inc., 874 F.2d 804, 10 USPQ2d 1843 (Fed. Cir.), cert. denied, 493 U.S. 975 (1989); In re Kulling, 897 F.2d 1147, 14 USPQ2d 1056 (Fed. Cir. 1990); and In re Geisler, 116 F.3d 1465, 43 USPQ2d 1362 (Fed. Cir. 1997)."
Therefore, the difference between the prior art and the invention encompasses wherein the polymer crosslinkers are a,b-unsaturated aldehyde polymers (e.g. see claim 25).
However, Buffa teaches hydrogels are routinely formed using aldehyde precursors and subsequent conjugation with biologically active N-nucleophiles or with polyamines and that these are one of the most versatile precursors (see page 293, introduction; Table 1; and section 2.9). Buffa teaches that the main advantage of the aldehydic groups is their ability to react with a wide range of amino compounds even under physiological conditions and for biocompatible hydrogels wherein in vivo crosslinking reactions are needed (see page 293-294, introduction). Buffa teaches the materials are compatible with optical analysis (see page 294, introduction) and yield enhanced properties thereof (see sections 3.5 and 4; and Table 3).
Therefore, it would have been prima facie obvious, before the effective filing date of the claimed invention, to a person of ordinary skill in the art, to modify the methods for preparing a biological sample for expansion microscopy, as taught by Boyden, by cross-linking the hydrogel with a,b-unsaturated aldehyde polymers, thereby arriving at the claimed invention, to produce a biocompatible hydrogel with enhanced properties, as taught by Buffa. Therefore, each and every element is taught in the prior art and the combination has a beneficial result. However, the combination amounts to no more than a predictable use of prior art elements according to their established functions. The person of ordinary skill in the art would have been motivated to make the modification, with at least a reasonable expectation of success because such hydrogels with already shown to be compatible with optical analyses, as taught by Buffa. Therefore, the combination leads to expected results because each element performs the same function as it does individually. With regards to the specific concentrations in dependent claim 23 and 24; it is also noted that MPEP 2144.05 states, generally, differences in concentration will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration 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).
Additionally, KSR International Co. v. Teleflex Inc., 127 S. Ct. 1727, 1741 (2007), discloses that combining prior art elements according to known methods to yield predictable results, is obvious unless its application is beyond that person's skill. KSR International Co. v. Teleflex Inc., 127 S. Ct. 1727, 1741 (2007) also discloses that the combination of familiar elements according to known methods is likely to be obvious when it does no more than yield predictable results. In the instant case, all elements (i.e. methods for preparing biological samples for expansion microscopy using cross-linked hydrogels and hydrogels cross-linked with a,b unsaturated aldehydes used in optical applications) were known in the art. In addition, combining these elements yields a method wherein each element merely performs the same function as it does separately; thus the results of the combination would be recognized as predictable to one of ordinary skill in the art. Therefore, it would have been obvious to a person of ordinary skill in the art to combine these prior art elements according to a known method to yield predictable results.
Therefore, the claimed invention is prima facie obvious in view of the teachings of the prior art, absent any convincing evidence to the contrary.
Applicant’s Arguments and Response to Arguments
17. All of Applicant’s arguments have been considered but were not deemed persuasive; accordingly, the rejection is maintained for reasons of record. For example:
With regards to the argument that the Office failed to establish a prima facie case of obviousness because the combination of references fails to teach bulk labeling (i.e. Buffa does not cure the deficiencies of Boyden; see Remarks, pages 9-10), the Office disagrees (see above) and reminds Applicant the combination was based on the limitation regarding the use of a,b-unsaturated aldehyde polymers, which the combination teaches. Thus, this argument is not persuasive.
Therefore, all of Applicant’s arguments have been considered but were not deemed persuasive; accordingly, the rejection is maintained for reasons of record.
New Rejection: Claim Rejections - 35 USC § 103
18. Claims 1-6, 8-17, and 19 are rejected under 35 U.S.C. 103 as being unpatentable over Tillberg et al. 2017 (WO 2017/027368) in view of Kozma et al. 2019 (Fluorogenic probes for super-resolution microscopy; Organic and Biomolecular Chemistry; 17:215).
Tillberg teaches methods for expansion microscopy (i.e. called proExM) comprising preparing samples for imaging, including the steps of chemical fixation, treatment with AcX (i.e. 0.01%; see page 24), gelation, digestion, and expansion in water, such that proteins are anchored to a swellable gel having cross-linking molecules are physically expanded by at least a factor of two in at least one dimension (e.g. see pages 4-5, bridging section; page 8, final paragraph; page 9, lines 5-12; and Figure 7; meeting limitations found in instant claims 1, 3, 17, and 19). Tillberg teaches the samples may be labeled post-expansion with a detectable label, including fluorescent molecules (e.g. see page 10, lines 14-17; Figure 8; and page 16, lines 6-21; meeting limitations found in instant claims 1 and 5). Tillberg teaches the use of more than one label each with a particular or distinguishable fluorescent property (e.g. see page 16, lines 23-24; meeting limitations found in instant claim 11). Tillberg teaches the use of bifunctional crosslinkers comprising protein reactive and gel reactive groups, including NHS-esters, thiols, amines, maleimides, and isocyanates (e.g. see page 9, lines 13-32; meeting limitations found in instant claims 9 and 10). Tillberg teaches the methods work with confocal microscopy and for a variety of sample types including HeLa cells and tissues, including mouse brain tissue (e.g. page 12, lines 19-26; page 13, lines 10-20; page 16, lines 1-5; meeting limitations found in instant claims 2, 4, 14, and 15-16). Tillberg teaches the HeLa cells were stain specifically for tubulin, clathrin and keratin (e.g. page 12, lines 19-22; and Figure 4; meeting limitations found in instant claims 8 and 14). Tillberg teaches the method provides optional steps for amplification (e.g. page 19, lines 12-13; meeting limitations found in instant claim 6) and automation (e.g. page 24, lines 10-14; meeting limitations found in instant claim 12). Tillberg teaches the embedded samples is expanded 3 to 100-fold or more (e.g. see page 19, lines 2-5; meeting limitations found in instant claim 13).
The difference between the prior art and the invention is that Tillberg are silent with regards to bulk labeling amino acids of non-identical molecules in newly amended independent claim 1.
However, Tillberg teaches that samples processed with proExM are optically clear and index-matched to water thereby allowing for super-resolution imaging deep into the sample using conventional fluorescent microscopes (e.g. page 14, lines 42-34) and teaches their invention enables the use of probes (e.g. page 15, lines 14-15).
In addition, Kozma teaches several fluorescent probes are useful with super-resolution imaging (e.g. see title and abstract) including their small size, greater flexibility and better photophysical properties such as a wider spectral range, greater photostability, and higher brightness (e.g. see introduction). Kozma teaches synthetic fluorophores can be applied to virtually any biomolecular structure (identical and non-identical) in contrast to other tags that are often limited to terminal labeling of proteins (e.g. see introduction). Kozma teach another advantage of the use of fluorophores is that they preserve the biological function of a protein that would otherwise be disrupted by using antibody binding (e.g. page 220, right column). Kozma provide multiple examples of probes, including the use of xanthene dyes for protein labeling, having favorable characteristics for bioimaging such as high water solubility, fluorescence quantum yields, and molar extinction coefficients (e.g. see page 216, left column).
Therefore, it would have been prima facie obvious, before the effective filing date of the claimed invention, to a person of ordinary skill in the art, to modify the strategy for labeling in the proExM methods as taught by Tillberg, by using one or more fluorogenic probes such as xanthene dyes, thereby arriving at the claimed invention, because samples processed via proExM were optically clear and index-matched to water to facilitate super-resolution imaging, as taught by Tillberg; and fluorogenic probes for super-resolution imaging (e.g. xanthene dyes) were recognized as having favorable characteristics labeling proteins for bioimaging including high water solubility, fluorescence quantum yields, and molar extinction coefficients, as taught by Kozma. Therefore, each and every element is taught in the prior art and the combination has a beneficial result; however, the combination amounts to no more than a predictable use of prior art elements according to their established functions. The person of ordinary skill in the art would have been motivated to make the modification because fluorescent probes were considered useful in super-resolution microscopy based on their small size and their ability to preserve the biological function of proteins that would otherwise be disrupted by using antibody binding and the samples processed via proExM were optically clear and indexed matched to water. The person of ordinary skill in the art would have had a reasonable expectation of success because Tillberg had already taught methods for the physical expansion of biological samples, including procedures for labeling the sample after expansion, had already taught the samples were designed to facilitate super-resolution microscopy processing, and had already taught their method enabled the use of probes; while Kozma had already demonstrated the advantages of multiple different types of fluorescent probes for use in super resolution microscopy. Therefore, the combination leads to expected results because each element performs the same function as it does individually.
Additionally, KSR International Co. v. Teleflex Inc., 127 S. Ct. 1727, 1741 (2007), discloses that combining prior art elements according to known methods to yield predictable results, is obvious unless its application is beyond that person's skill. KSR International Co. v. Teleflex Inc., 127 S. Ct. 1727, 1741 (2007) also discloses that the combination of familiar elements according to known methods is likely to be obvious when it does no more than yield predictable results. In the instant case, all elements (i.e. methods for expansion microscopy of samples to facilitate imaging; and methods for super resolution microscopy for labeling proteins for imaging and using bulk labels for multiple non-identical proteins) were known in the art. In addition, combining these elements yields a method wherein each element merely performs the same function as it does separately; thus the results of the combination would be recognized as predictable to one of ordinary skill in the art. Therefore, it would have been obvious to a person of ordinary skill in the art to combine these prior art elements according to a known method to yield predictable results. Consequently, the claimed invention is prima facie obvious in view of the teachings of the prior art, absent any convincing evidence to the contrary.
Conclusion
19. No claims are allowed.
20. Any inquiry concerning this communication or earlier communications from the examiner should be directed to MARY MAILLE LYONS whose telephone number is (571)272-2966. The examiner can normally be reached on Monday-Friday 8 am to 5 pm EST. Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http: //www.uspto.gov/interviewpractice. If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Dan Kolker can be reached on (571)-272-3181. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
21. Information regarding the status of an application may be obtained from the Patent Application Information Retrieval (PAIR) system. Status information for published applications may be obtained from either Private PAIR or Public PAIR. Status information for unpublished applications is available through Private PAIR only. For more information about the PAIR system, see http://pair-direct.uspto.gov. Should you have questions on access to the Private PAIR system, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative or access to the automated information system, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/MARY MAILLE LYONS/Examiner, Art Unit 1645
January 14, 2026