US 11333588

I'll run several targeted searches — one for the patent itself, one aimed at USPTO records, and one at CAFC dockets.

Let me verify the granted claim set and check for any CAFC/appeal activity specifically.

Summary of US Patent 11,333,588 B1

Search scope note: I searched general web indexes that surface USPTO/Google Patents/Justia/uspto.report records and looked for Federal Circuit (CAFC) appellate activity. I found no CAFC docket, appeal, IPR, or PTAB proceeding associated with this patent. The only non-patent hit for the literal string "11333588" was a Brazilian state commercial-registry notice (JUCISRS) listing "LC IN DUSTRA DE EXAUSTORES LTDA" — that is a company-registration number, not this patent and not a court case. Treat it as noise. I could not access a dedicated CAFC docket database directly, so I cannot certify with certainty that no appeal exists; I state only that no such activity appeared in the sources I could reach.

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Bibliographic data (all consistent across the patent PDF, Google Patents, and Justia)

Abstract (as printed)

"Matrix-assisted methods and compositions, including those based on solutions containing low melting agarose, to prepare intact organs and other samples for super resolution imaging by microscopy, and more particularly, lightsheet microscopy."

(The fetched abstract text was truncated mid-word at "super res…"; the full sentence above is reconstructed from the patent front page and matches the Google Patents listing.)

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Plain-language overview of the claims

Important distinction: the patent body contains a long numbered list of "Embodiments 1–161" (e.g., "Embodiment 34…"). These are specification embodiments, not claims. Several are broader than the granted claims. The granted claim set is separate and independent of that list. This is a frequent source of confusion when reading this document.

Claim 1 is the sole independent claim. In the claim set as rendered by Justia, every one of claims 2–26 depends (directly or indirectly) on claim 1. So the enforceable scope rises or falls on claim 1.

Claim 1 (independent) — plain language: A method of preparing an expanded biological sample with four steps:

• (a) pre-incubate a biological sample that is a cell, tissue or organ in a gelling solution containing a hydrogel precursor;
• (b) add a polymerization initiator to the gelling solution and polymerize it, so the sample becomes embedded in a hydrogel;
• (c) homogenize the hydrogel-embedded sample in a denaturing solution — i.e., the homogenization must be by denaturation (not, e.g., enzymatic digestion) — to denature the sample; and
• (d) incubate the denatured, hydrogel-embedded sample in an expansion solution that is an aqueous solution of from 0.1% to 1% low-melting-point (LM) agarose, causing the sample to expand.

The inventive hook is step (d): using dilute LM agarose in the expansion/swelling liquid as a supporting matrix so that expanded (otherwise fragile, water-rich) samples can be handled, sliced and imaged. Note that claim 1 requires the 0.1%–1% range and requires denaturation-based homogenization — both are narrower than broad statements in the specification.

Dependent claims, grouped by theme (all narrower than claim 1):

• LM agarose concentration: 0.3%–0.6% (claim 2).
• Expansion conditions: ≥37 °C (claim 3); at least two days (claim 4).
• Expansion solution with staining: dye/label/stain added to the expansion solution (claim 5), specifically lectin, DAPI, propidium iodide, acridine orange, or combinations (claim 6) — i.e., staining during expansion.
• Imaging add-ons: high-resolution imaging (claim 7); transfer to water before imaging (claim 8); mount on holder and image in a water chamber (claim 9); embed in an LM agarose block (claim 10); the scan-and-slice workflow — image a first layer, remove it, image the adjacent second layer (claim 11).
• Homogenization chemistry: denaturing solution comprises an enzyme or a detergent (claim 12).
• Sample identity: rodent organ (claim 13); organ (claim 14); whole organ (claim 15); brain (16), kidney (17), liver (18), lung (19), spleen (20), intestine (21), stomach (22); tissue (23); brain tissue (24); hippocampus (25); hypothalamus (26).

Bottom line: this is a single-independent-claim patent to a four-step expansion-microscopy workflow distinguished by swelling the hydrogel-embedded sample in a 0.1–1% low-melting agarose bath, with dependent claims capturing staining-during-expansion, whole-organ specimens, and the agarose-block scan-and-slice imaging technique.

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Points of uncertainty / caution

1. Claim-set rendering. My claim text comes from the Justia rendering of the patent (and is corroborated by the front-page "26 Claims" count). The Google Patents text supplied in the case file lists only the specification "Embodiments," not the granted claims, so I could not cross-check the claims against the authoritative granted text in that document itself. The claim language above should be verified against the USPTO PatentCenter/Patent Public Search certified copy before being relied on for an infringement or validity opinion.
2. Single independent claim. I am reasonably but not absolutely confident that claim 1 is the only independent claim, based on the dependency structure as rendered. If the rendering silently omitted a claim or merged an independent claim, that conclusion would change.
3. No litigation/appeal found. Absence of CAFC/PTAB hits in my searches is not proof of absence. A definitive check requires the CAFC docket (PACER/CM-ECF or the CAFC's own docket search) and PTAB's P-TACTS/AIAPI, neither of which I could query directly here.
4. Minor internal inconsistencies in the patent text (unchanged, per the literal-interpretation rule): the specification variously recites "200 nM Boric Acid" in Embodiment 10/54/130 versus "200 mM Boric acid" in Example 1/Table 1; and the transducer name appears as "Na+K+Cl— Cotransporter 2 (NKCC2)." The granted claim 1 does not depend on these values, so they do not affect claim scope but do matter if you are relying on the examples for written-description or enablement analysis.
5. Assignee identity. "Nebulum Technologies Co., Ltd." (Zhubei, TW) is distinct from an unrelated California entity, "Nebulum Technologies, LLC." Do not conflate them.

I'll research litigation involving US Patent 11333588 using the specified sources.

Let me search more specifically for the patent owner and related litigation.

Let me try additional targeted searches to be thorough.

Litigation Search Report — U.S. Patent No. 11,333,588

Patent identified (literal match only): U.S. Patent No. 11,333,588 B1 — "Matrix-assisted methods and compositions to prepare biological samples for super-resolution imaging" (Appl. No. 17/113,873; filed 2020-12-07; granted 2022-05-17; assignee Nebulum Technologies Co., Ltd., Zhubei, TW).

Finding: No litigation located

Based on the searches I was able to run, I found no litigation — district court, ITC § 337, PTAB (IPR/PGR), or CAFC — in which U.S. Patent No. 11,333,588 is (or was) asserted or challenged. I cannot confirm this is an exhaustive negative; see caveats below.

Evidence reviewed and why it does not support a hit

No plaintiff, defendant, jurisdiction, case number, filing date, or outcome can therefore be reported, because no qualifying case was identified.

Caveats (stated explicitly, per search-integrity requirement)

1. Search scope limitation. I could not directly query PACER, the Unified Patents litigation portal (portal.unifiedpatents.com/litigation/caselist), Docket Alarm, or Lex Machina in this session; findings rest on general web searches. A docket-level check is advisable before treating this as a definitive negative.
2. Patent is recent and narrowly held. It issued 2022-05-17 to a small Taiwan-based assignee (Nebulum Technologies Co., Ltd.). A negative result is plausible but not certain.
3. Number-conflation risk. Several real, active cases involve similar-looking but distinct numbers ('884, '885, '335, '386, 11,813,388, 11,465,005, 10,561,884, 10,799,745). None of these is U.S. 11,333,588, and I have not attributed any of them to this patent.
4. No auto-correction applied. I treated "11333588" literally as U.S. Patent No. 11,333,588 B1 and excluded the Japanese utility model JP 11333588 U and the Brazilian registry entry.

Bottom line: On the record available to me, no known litigation involves U.S. Patent No. 11,333,588. If you need a court-verified negative, the next step is a direct PACER/Unified Patents docket query on patent number 11333588 and on assignee "Nebulum Technologies Co., Ltd."

I'll verify the structured-data default with targeted searches before writing anything up.

PTAB Activity Report — U.S. Patent No. 11,333,588 ("Matrix-assisted methods and compositions to prepare biological samples for super-resolution imaging")

Prepared: 2026-09-29 · Patentee: Nebulum Technologies Co., Ltd. (Zhubei, TW) · Inventors: Tian, Lee, Chen · Appl. No.: 17/113,873 · Filed: 2020-12-07 · Granted: 2022-05-17 · Anticipated expiration: 2040-12-07 · Claims: 26

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Proceedings overview

There are zero AIA trial proceedings on file against US 11,333,588 — no IPR, no PGR, no CBM — which means the patent is entirely UNTESTED at the PTAB: no claims have been canceled, no claims have been judicially sustained, no institution decision exists, and there is no § 315(e)(2) estoppel on the books, leaving a defendant a clean slate to file the first petition with any art it chooses.

The structured "PTAB proceedings on file" block supplied with this task reports that the USPTO Open Data Portal returns no AIA trial proceedings for this patent as of the most recent ingest. My independent searches (see "Search record" below) surfaced no IPR/PGR/CBM number, no PTAB Final Written Decision, and no Federal Circuit appeal naming this patent or this patentee. I therefore am not populating the per-proceeding template — there is no proceeding to describe, and inventing one would be fabrication.

A caution about the instruction framing: the task prompts assumed a scenario such as "the patent has survived two IPRs and is hardened" or "claims 1–5 have been canceled." Neither is true here. The record is simply empty. Do not let a demand letter or an internal summary tell you otherwise without a PTAB number to check.

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Why the record is empty (and what it does and does not mean)

• No litigation found either. I found no U.S. district court action (D. Del., E.D./W.D. Tex., N.D. Cal., etc.) in which Nebulum Technologies is the plaintiff asserting the '588 patent. No IPR typically follows from no litigation — PTAB challenges are overwhelmingly filed by accused infringers or their indemnitors.
• Commercial footprint is small. Public assignee/WIPO-style records describe Nebulum as a single-patent-portfolio, single-grant entity as of 2022-05-17. A patentee with no U.S. enforcement campaign gives sophisticated defendants no reason to spend $400k–$800k on a petition.
• The absence is a signal, but a weak one. Well-asserted patents eventually attract IPRs; a never-asserted patent frequently does not. The realistic reading is "not yet in play," not "validated." This patent is a plausible future assertion vehicle — expansion microscopy is commercially active (clearing/reagent vendors, light-sheet imaging platforms, spatial-omics toolmakers), and the patent's claimed subject matter (LM agarose in the expansion solution) is a reagent-level, easy-to-detect practice.
• No ex parte reexamination activity surfaced in my searches either. That is a secondary check, not a substitute for a PTAB E2E / Patent Center query.

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What the patent actually claims (needed for any defensive analysis)

Because there is no FWD to quote, the operative claim scope is the granted text. Independent claim 1 (as printed in the patent) recites, in substance:

A method of expanding a biological sample, comprising: (a) a gelling solution; (b) introducing a polymerization initiator to the gelling solution and polymerizing the gelling solution to embed the biological sample in a hydrogel; (c) homogenizing the biological sample embedded in the hydrogel in a denaturing solution to denature the biological sample; and (d) incubating the denatured biological sample embedded in the hydrogel in an expansion solution comprising an aqueous solution of from 0.1% to 1% low melting point (LM) agarose.

Claims 2–26 are dependents: 0.3–0.6% LM agarose (cl. 2), ≥37 °C (cl. 3), ≥2 days (cl. 4), dye/stain (cl. 5–6), imaging steps (cl. 7–11), enzyme-or-detergent denaturation (cl. 12), and organ/tissue species claims (cl. 13–26: brain, kidney, liver, lung, spleen, intestine, stomach, hippocampus, hypothalamus).

Key defensive observations on the face of the claims:

1. Every claim requires LM agarose in the expansion solution. This is a narrow, additive limitation over stock ExM/proExM/MAP protocols. Prior art that expands a hydrogel in plain water, PBS, or standard agarose does not anticipate claim 1. A § 103 case must supply the motivation to put LM agarose in the swelling bath — which is exactly what the specification frames as the invention.
2. Claim 1 requires denaturing-type homogenization, not protease digestion. Art teaching proteinase-K homogenization alone is one element short of claim 1 as granted (though such art is directly relevant to claims drawn to digestion).
3. The patent's own cited art is the natural starting point, and the applicant put much of it on the face of the patent: Chen et al., Expansion Microscopy, Science 347(6221):543–548 (2015); Tillberg et al., Nat. Biotechnol. 34:987–992 (2016) (proExM); Ku et al. (2016) (MAP); Klimas et al., Curr. Protoc. Cytom. 91(1):e67 (2019); Gao et al., BMC Biology 15(50) (2017); Asano et al., Curr. Protoc. Cell Biol. 80(1) (2019); Murakami et al., Nat. Neurosci. 21:625–637 (2018) (CUBIC-X); Chung et al., Nature 497:332–337 (2012) (CLARITY); Ertürk et al., Nat. Protoc. 7:1983–1995 (2012) (3DISCO); Susaki et al., Cell 157:726–739 (2014) (CUBIC). These were before the Examiner; a § 325(d) argument by the patentee would have real traction unless new art is materially different, so a petition should lead with art that was not of record.
4. Priority/filing date: 2020-12-07, with no earlier priority claim shown. That date is the § 102 cut-off for printed publications and patents, and it is relatively late for this field (ExM dates to 2015). There is a plausible § 102(a)(1) public-use / on-sale / printed-publication line of inquiry into the inventors' own pre-2020-12-07 disclosures (Bi-Chang Chen's lab is at Academia Sinica, a prolific publisher and conference presenter), but I have not verified any specific pre-filing disclosure and will not assert one. If you are evaluating a petition, this is the highest-value first search.

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Strategic summary

Claim status: all 26 claims are UNTESTED. There is no canceled claim, no surviving-as-narrowed claim, and no adjudicated claim. Any statement that "claim 1 is dead" or that "the patent has survived IPR" is unsupported by the record. The full claim set — independent claim 1 plus dependents 2–26 — remains live and enforceable, subject only to ordinary validity defenses in whatever forum you find yourself.

Estoppel landscape: none applies. Because no IPR/PGR was ever instituted (or even filed), no petitioner is barred under 35 U.S.C. § 315(e)(1) or (e)(2). For a defendant now facing assertion, this is a genuine advantage: you may choose your own art, your own expert, and your own claim-construction positions without working around a prior panel's findings. The flip side is informational — there is no Board claim construction, no institution decision explaining what "low melting point agarose" or "homogenizing ... in a denaturing solution" means, and no FWD to mine. You will be building the record from zero. Note also the § 315(b) one-year clock runs from service of a complaint alleging infringement; if you are served, calendar that date immediately, because a § 315(b)-barred petition cannot be cured.

Pattern signals: no repeat-petitioner pattern is observable (there is no petitioner). I found no evidence of a defensive aggregator (Unified Patents, RPX, or similar) challenging this patent, and no PTAB or Federal Circuit appellate activity by this patentee. Nebulum does appear to be an active filer beyond the '588 patent — for example, EP 4067865 appears associated with Nebulum Technologies in a 2024 Netherlands IP bulletin (the bulletin entry is garbled as to which title maps to which proprietor, so verify before relying on it), and a related Nebulum publication US 2024/0174818 A1 exists. If the '588 patent is ever asserted, expect sibling/continuation patents to be asserted alongside it, which materially changes the economics of a single-patent IPR. A related-family sweep and a continuation-status check on the 17/113,873 family are the first diligence items, not the IPR itself.

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Recommended next steps

If you are a defendant and were hoping to lean on an existing PTAB outcome: there is none to lean on. Do not cite a nonexistent FWD, and demand the PTAB number from anyone who tells you one exists. No claim disposition to quote, because no Final Written Decision has issued.

What to do instead:

1. Confirm the empty record yourself, today, at the source: run the patent number in PTAB E2E / PTAB Patent Search — https://ptacts.uspto.gov/ptacts/ — and cross-check assignment and transaction history in USPTO Patent Center. Five minutes of primary-source verification beats any secondary summary, including this one. For any proceeding number you do find, the canonical public links are the PTAB E2E documents tab and, for appeals, the Federal Circuit docket and CourtListener.
2. Run a family sweep before drafting anything. Identify continuations/divisionals of 17/113,873 and Nebulum's EP/other foreign counterparts. A validity opinion on the '588 patent alone is not a freedom-to-operate answer if there are three siblings.
3. Front-load the pre-December-2020 prior-art search. Given the 2015–2019 ExM literature base, the winning references are likely the ones not on the face of the patent: (a) any publication using low-melting/low-gelling-temperature agarose as an embedding or support matrix for expanded or cleared tissue; (b) any pre-2020-12-07 disclosure by the inventors themselves (papers, preprints, conference abstracts, theses, grant narratives). Because the applicant's own cited art is on the record, § 325(d) discretion is a live risk — lead with new art.
4. Preserve the option value. With no estoppel and no prior Board constructions, you can (i) file IPR within one year of service with your best art, (ii) rely on district-court invalidity, and (iii) consider ex parte reexamination or a § 112 written-description/enablement attack on the organ-scope dependents (claims 13–22, 23–26 cover an enormous range of whole organs and tissue types from a mouse-organ working example — that breadth is worth a hard look, but it is an argument, not a finding).
5. If you are the patentee or evaluating acquisition: the same emptiness cuts the other way — the patent has never been stress-tested, its narrow LM-agarose limitation is the only thing separating it from the pre-2015 ExM literature, and its broad organ/tissue dependents are the most vulnerable claims. Value the asset accordingly.

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Search record (for reproducibility)

Searches run 2026-09-29 on: US11333588 IPR PTAB Nebulum Technologies; "Nebulum Technologies" patent inter partes review PTAB; "11333588" patent litigation OR IPR OR "inter partes review"; "Nebulum Technologies" lawsuit patent infringement expansion microscopy; ptacts.uspto.gov "11333588" OR "11,333,588"; and Nebulum Technologies courtlistener docket patent case plaintiff. Results returned only the patent's own Google Patents/Justia pages, unrelated TPAB petitions on other parties' patents, and non-patent hits on the string "11333588" (a Brazilian corporate filing, a Chinese trademark registration, a UK company number, and a 1988 Japanese utility model). No AIA trial proceeding, no PTAB decision, and no Federal Circuit appeal involving US 11,333,588 or Nebulum Technologies was located.

A note on scope and confidence: I am highly confident in the negative finding for AIA trial proceedings, because it is corroborated by the structured ODP data plus multiple independent searches. I am not confident that no district court action exists — my searches did not include a full PACER or Docket Navigator sweep, and pleadings in a freshly filed case may not yet be indexed by general web search. Treat the litigation-absence statement as "nothing found," not "nothing exists."

Primary references: US 11,333,588 on Google Patents · granted claim text (Justia) · PTAB E2E / Patent Search · CourtListener

I'll research the assignment record for US 11333588 before assembling the analysis.

Let me dig for the specific assignment record details.

Ownership & Assignment Analysis — US 11,333,588 B1

Patent: US 11,333,588 B1 ("Matrix-assisted methods and compositions to prepare biological samples for super-resolution imaging")
Application: 17/113,873 · filed 2020-12-07 · granted 2022-05-17 · anticipated expiration 2040-12-07
Source of record below: Google Patents legal-events feed for US11333588B1 (fetched 2026-09-24) plus public registry data on the assignee.

Tooling disclosure / caveat. I was unable to open the USPTO Assignment Center (https://assignmentcenter.uspto.gov/) or the legacy AOTW index (https://assignment.uspto.gov/patent/index.html) in this session, so I could not retrieve or verify reel/frame numbers, execution dates, or the correspondent of record. Everything in the timeline below is drawn from the Google Patents legal-events feed, which surfaces assignee/assignor names but not reel/frame or correspondent. I have not invented any reel/frame values. Where a fact is unverified I say so.

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Inventors

Notes / unusual patterns:

• The Google Patents legal-events entry for the recorded assignment lists assignors as "LEE, CHIA-MING, TIAN, XUEJIAO" only — the third named inventor, Bi-Chang Chen, does not appear among the assignors in that record. This is either (a) a truncation/incomplete rendering of a multi-page assignment, or (b) evidence of a separate assignment record for Chen, which is common when a co-inventor's rights are held or co-owned by an academic employer. This needs to be confirmed against the actual reel/frame documents — I could not verify it.
• No evidence was found that any inventor departed the original assignee within 12 months of filing. This pattern is not observable from the data available. Nebulum was a brand-new entity (incorporated 2020-05-20, ~7 months before filing), which is itself notable: the patent was filed essentially at the company's inception.
• I could not confirm employment relationships for any inventor. Bi-Chang Chen is plausibly affiliated with an academic institution (his name coincides with a known lattice light-sheet microscopy researcher), but this is an unverified inference and should not be treated as a finding. Note that the "Chen et al., Expansion Microscopy, Science 347(6221), 2015" reference cited on the face of the patent is Fei Chen (MIT/Boyden lab), a different person from inventor Bi-Chang Chen — do not conflate them.

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Original assignee

Nebulum Technologies Co., Ltd. (Chinese name: 諾倫科技股份有限公司; Taiwan company ID 83761687)

• Named on the issued patent at (71) Applicant and (73) Assignee: "Nebulum Technologies Co., Ltd., Zhubei (TW)."
• Primary line of business: biomedical imaging services — tissue clearing, whole-tissue immunolabeling, light-sheet microscopy, 3D image analysis. Per company profiles, it is a service provider in the biotechnology sector rather than a manufacturer of instruments. Registered business items include optical instrument manufacturing and intellectual property rights (F601010 智慧財產權業), among others.
• Does it ship a product embodying the claims? It commercializes the patented method as a 3D whole-organ imaging service (per public seminar materials from Taiwan's National Health Research Institutes, NHRI, dated 2021-01-21 and 2022-02-17, presented by CEO Billy Huang / 黃元孝). It is a method claim patent, so the claims read on the process Nebulum performs, not on a physical article sold.
• Corporate status: Operating. Taiwanese registry data shows the company is "營業中" (in business), with paid-in capital growing from NT$12M (2020) to ~NT$59.9M (2024) to ~NT$79.4M (2025), and registered capital raised to NT$300M by April 2026. It is a closed company (閉鎖性) under Taiwan law. Registered address moved from Zhubei, Hsinchu County → Nangang, Taipei City in November 2021. Officers: 黃元孝 (Chairman/CEO, majority holder), 黃仲傑 (Supervisor since Sept 2024, replacing 陳仕傑). ~1–10 employees. Estimated revenue <US$1M. Privately held; no subsidiaries reported.
• Not acquired, dissolved, or in bankruptcy as of the latest registry data available.

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Assignment timeline

Only one recorded assignment appears in the chain of title as reflected in the Google Patents legal-events feed. No post-issuance assignments were found.

• Executed: not shown in the source consulted / recorded 2020-12-08 — Reel not retrievable in this session (Assignment Center inaccessible)
  • Conveyance: ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS).
  • Assignor: LEE, CHIA-MING; TIAN, XUEJIAO (note: third inventor Bi-Chang Chen is absent from this record as rendered — see Inventors section)
  • Assignee: NEBULUM TECHNOLOGIES CO., LTD.
  • Correspondent: not retrievable. Because the Assignment Center was unreachable in this session, I cannot name the attorney/firm of record. I am expressly declining to guess — this is the single most important field for the repeat-player analysis and it is unverified.
  • Context: routine founder/inventor-to-company assignment at formation, filed one day after the application (2020-12-07). This is a standard "assignment on filing" record, not an acquisition, fire-sale, reorg, or securitization.

If the Assignment Center genuinely holds no records beyond this one — the more likely reading given Nebulum is a 2020 startup that still owns the patent — then the original assignee still owns US 11,333,588. That is a substantive finding in itself: no NPE acquisition chain exists on the public record.

Verification link: search patent number 11333588 at USPTO Assignment Center (or the legacy AOTW) to confirm reel/frame, execution date, and correspondent.

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Timeline diagram

timeline
    title Ownership of US 11333588
    2020 : Company founded in Zhubei Taiwan
         : Application filed 7 Dec
         : Inventors assign rights to Nebulum
    2022 : Patent issued 17 May
    2021 : HQ relocated to Nangang Taipei

(The 2021 relocation row is placed after 2022 to keep the diagram readable; chronologically it falls between the 2020 and 2022 events.)

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NPE / troll-pattern signals

1. Shell-entity transfer — Not present. No transfer out of the operating assignee appears on the record. Nebulum is a going concern with an active corporate registration, growing paid-in capital, a named CEO, a public-facing service business, and public seminar activity — the opposite of a single-purpose anonymous LLC. There is no "IP / Holdings / Ventures" successor in the chain.

2. Known asserter in the chain — Not present. Neither Nebulum Technologies Co., Ltd. nor any recorded assignee matches the named NPE lists (Acacia, Marathon, IV, IPNav, Wi-LAN, Mosaid/Conversant, Vringo, Pendrell, MPHJ, Lumen View, Round Rock, DGC, Spangenberg entities). No Unified Patents or RPX assertion record was surfaced for this patent. Caveat: I was unable to query the RPX/Unified asserter directories directly in this session, so this is "not found," not "affirmatively excluded."

3. Repeat correspondent across the chain — Unclear / not assessable. With only one link in the chain and the Assignment Center unreachable, there is no recurrence to measure. The correspondent of record is the key unknown in this analysis and must be pulled from the reel/frame before any repeat-player call can be made. No finding.

4. Cascading transfers — Not present. One recorded link, executed at company formation. No chained LLCs, no shared correspondent addresses, no common principals across successive assignees.

5. Pre-litigation transfer — Not present. No infringement suit naming US 11,333,588 was surfaced. With no litigation, there is no trigger event to back-date against. Caveat: I could not complete a docket sweep (PACER/Docket Alarm) before the tool limit; absence of a surfaced suit is not proof none exists.

6. Bankruptcy fire-sale — Not present. No Chapter 7/11 record for Nebulum; registry status is active/operating.

7. Privateering — Not present. No operating company → NPE transfer, and no SEC filing evidence (Nebulum is private and not a US registrant; no 10-K/8-K trail exists).

8. Defensive aggregator (anti-NPE) — Not present. The chain does not terminate at RPX, AST, LOT, Unified, or OIN. Nebulum is a commercial operating entity, not a defensive aggregator.

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Verdict

Insufficient data (only the original inventor-to-company assignment is on the public record)

Justification: The only ownership event visible — recorded 2020-12-08, assignors Lee and Tian to Nebulum Technologies Co., Ltd., one day after the 2020-12-07 filing — is a routine formation-stage inventor assignment to an operating company, and no subsequent transfer, licensing vehicle, or asserting entity appears in the chain. None of the eight NPE signals is affirmatively present; the entity in title is a funded, revenue-generating Taiwan imaging-services startup that practices the claimed method commercially. Because the Assignment Center was unreachable, I could not verify reel/frame, execution date, the correspondent of record, or whether Bi-Chang Chen was separately assigned — those three gaps are the residual uncertainty. Re-run this analysis after pulling the reel/frame at assignmentcenter.uspto.gov for patent 11333588, and in particular capture the correspondent field, which is the only field capable of converting this to a firm finding in either direction.

I'll research US 11333588 and its cited prior art.

Let me get the specific citation list and examine each reference.

Let me pull the patent's own "References Cited" section and the full citation tables.

1. Identification of the patent (literal reading of "11333588")

Sources: https://patents.google.com/patent/[US11333588B1](/patent/US11333588B1)/en ; https://patentimages.storage.googleapis.com/85/e1/e6/18d986c1423545/US11333588.pdf ; https://patents.justia.com/patent/11333588

Note on dates: the task header states the current date is 26 April 2026, while the operating system says 29 September 2026. I have not "corrected" either; the analysis below uses the stated task date of 26 April 2026, which does not change any conclusion since the patent granted on 17 May 2022.

Note on scope: I confined the search to US 11,333,588 / US 17/113,873. Numbers that appeared alongside it in family/citation tables (e.g., US 11,408,890; US 10,059,990; US 7,838,302 — all unrelated MIT/Harvard patents) are not US 11,333,588 and are excluded except where noted as context.

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2. The claim set that the prior art must be measured against

Claim 1 (the only independent claim; all of claims 2–26 depend from it):

1. A method of preparing an expanded biological sample, comprising:
   (a) pre-incubating a biological sample that is a cell, tissue or organ in a gelling solution, the gelling solution comprising a hydrogel precursor;
   (b) introducing a polymerization initiator to the gelling solution and polymerizing the gelling solution to embed the biological sample in a hydrogel;
   (c) homogenizing the biological sample embedded in the hydrogel in a denaturing solution to denature the biological sample; and
   (d) incubating the denatured biological sample embedded in the hydrogel in an expansion solution, to expand the biological sample, the expansion solution comprising an aqueous solution of from 0.1% to 1% low melting point (LM) agarose.

Claims 2–26 add: 0.3–0.6% LM agarose (2); ≥37 °C (3); ≥2 days (4); dye/label/stain (5); lectin/DAPI/PI/acridine orange (6); high-resolution imaging (7); transfer to water (8); water chamber/mount (9); LM agarose block (10); section-imaging-remove-imaging (11); denaturing solution = enzyme or detergent (12); rodent organ / organ / whole organ / brain / kidney / liver / lung / spleen / intestine / stomach (13–22); tissue / brain tissue / hippocampus / hypothalamus (23–26).

This dependency structure is decisive for the § 102 question: because every claim depends from claim 1, a reference can only anticipate the set if it discloses all of steps (a)–(d), including the 0.1–1% LM agarose expansion solution. That limitation is the point of novelty of the patent (the "matrix-assisted" concept, see the Definitions section: "the compositions comprise an expansion solution comprising agarose, and more particularly, low melting (LM) agarose, for expanding biological samples embedded in a swellable material").

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3. Patent citations appearing for US 11,333,588

3.1 The two references returned as the "Citations" table for this patent number

Sources: https://patents.google.com/patent/EP3539036A4/en (citation table listing both documents against US11333588B1); https://www.patents-review.com/a/20150087001-methods-phenotyping-intact-tissues.html ; https://patentimages.storage.googleapis.com/1f/63/11/d8f4de905214c5/[US10746981](/patent/US10746981).pdf

3.2 U.S. patent documents printed on the front page of the '588 patent

The OCR of the granted patent's "[56] References Cited / U.S. Patent Documents" block yields the following numbers, which I am reporting as printed (the OCR is partly truncated and I could not verify every title — this is stated explicitly rather than guessed):

• US 10,309,879 B2* (June 2019)
• US 2009/0041316 A1* (Feb 2009)
• US 2009/0142259 A1* (June 2009)
• US 2010/0075391 A1* (March 2010)
• US 2016/0258856 A1* (Sept 2016)
• US 2016/0305856 A1* (Oct 2016)
• US 2017/0199104 A1* (July 2017)
• US 2018/0030504 A1* (Feb 2018)
• US 2019/0106037 A1* (April 2019) — OCR reads "2019/006037"
• US 2019/0113423 A1* (May 2019)
• US 2019/0145868 A1* (Aug 2019) — date/number partially ambiguous in OCR

Source: https://patentimages.storage.googleapis.com/85/e1/e6/18d986c1423545/US11333588.pdf (§ (56))

Confidence flag: I could not verify the titles, assignees or subject matter of these eleven U.S. patent documents with high confidence, and I could not open the USPTO Patent Center "References Cited" (PTO-892) view directly. For at least one of them (US 10,309,879 B2) I have no reliable recollection of the subject matter, so I will not attribute a title. Any § 102 conclusion as to these documents should be re-verified against the patent's own front page.

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4. Non-patent literature cited on the front page of the '588 patent (titles verified from the OCR)

Additionally, the description of the '588 patent itself cites WO 2015/127183, WO 2017/027368, WO 2017/027367, WO 2017/147435, WO 2017/190101, WO 2019/156957, WO 2019/241662, WO 2020/013833 (ExM families), Tillberg et al. 2016, Nat. Biotechnol. 34:987–992 (proExM), Ku et al. 2016 (MAP), Chung et al. 2012, Nature 497:332–337 (CLARITY), Susaki et al. 2014, Cell 157:726–739 (CUBIC), Murakami et al. 2018, Nat. Neurosci. 21:625–637 (CUBIC-X), Ertürk et al. 2012, Nat. Protoc. 7:1983–1995 (3DISCO), Tainaka et al. 2018, Cell Reports 24:2196–2210, and Reveles Jensen and Berg, 2017, J. Chem. Neuroanatomy 86:19–34. These are applicant-acknowledged art and are candidates for a § 103 obviousness analysis even though they were not cited against the claims.

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5. § 102 anticipation analysis, reference by reference

The '588 patent is an AIA patent (filed 2020-12-07, no earlier priority), so §§ 102(a)(1) and 102(a)(2) apply; the critical date is 2020-12-07, and every item cited above published well before it.

Bottom line on § 102

None of the citations I was able to verify anticipates any of claims 1–26 of US 11,333,588. The reason is structural: every one of the 26 claims depends from claim 1, and claim 1 requires step (d) — expansion in an aqueous solution of 0.1% to 1% low melting point (LM) agarose. The cited art universally discloses the pre-expansion architecture (hydrogel embedding → denaturation/digestion) but either (i) expands in plain water (ExM/proExM), (ii) clears without expanding (CLARITY/PACT-type art such as US 2015/0144490 A1 and US 2015/0087001 A1), or (iii) uses LM agarose only after expansion as a mounting/sectioning block (Asano et al.; Strobl et al. for LSFM). The claimed concept — expanding the hydrogel-embedded, denatured specimen in the presence of an LM agarose matrix at 0.1–1%, so that the agarose infiltrates and stiffens the swelling gel — is not shown.

Where these references would instead matter:

• § 103 obviousness, not § 102: US 2015/0144490 A1 or US 2015/0087001 A1 (steps a–c) in view of Chen et al. 2015 (expansion microscopy) and Asano et al. 2019 or Strobl et al. 2017 (routine use of low-melting agarose with hydrogel/tissue specimens for handling and light-sheet imaging). The applicant's own specification frames the invention as solving a recognisable problem (expanded gels are "delicate," "soft and fragile," up to 99% water, and must be moved slowly or sliced), so a motivation-to-combine argument is available.
• Anticipation of a sub-combination only (not a claim): US 2015/0087001 A1 is the closest single reference for elements (a)–(c) because it expressly recites acrylamide at 1–20%, VA-044, and SDS at 1–30% — the same chemistries the '588 patent claims in embodiments 33/36–47 — but it does not reach claim 1's step (d).
• § 102 for dependent-claim subject matter in isolation: Mali et al. 1984 (acridine orange), Strobl et al. 2017 (LSFM of agarose-embedded specimens), Wong et al. (microtomy-assisted imaging of whole organs). None of these can anticipate a dependent claim because each dependent claim incorporates claim 1's LM-agarose limitation.

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6. Explicit limitations of this report

1. I could not retrieve the USPTO Patent Center / PTO-892 "References Cited" list directly; the citation data above come from the patent PDF front page (OCR), Google Patents, and Justia. The OCR of the U.S. patent-document block is partially truncated, so § 3.2 numbers should be treated as unverified leads.
2. The "Citations (2)" table returned for this patent number (US 2015/0087001 A1 and US 2015/0144490 A1) appeared in a Google Patents citation table associated with US11333588B1; because that table also appears on the EP 3539036 A4 page, there is a residual possibility that one or both documents are citations of a family member rather than of the '588 front page. Both are, in any event, relevant art on their face.
3. I have not fabricated titles or assignees for US 10,309,879 B2 or the other unverified U.S. publications. If you can supply the PTO-892 or the granted patent's front page image, I can complete the per-reference § 102 mapping.
4. This is a technical analysis, not a legal opinion; anticipation/obviousness determinations are ultimately the province of the USPTO or a court.

Primary sources used: https://patents.google.com/patent/US11333588B1/en · https://patentimages.storage.googleapis.com/85/e1/e6/18d986c1423545/US11333588.pdf · https://patents.justia.com/patent/11333588 · https://uspto.report/patent/grant/11,333,588 · https://www.patents-review.com/a/20150087001-methods-phenotyping-intact-tissues.html · https://patents.google.com/patent/EP3539036A4/en

I'll ground the obviousness analysis by checking the prior-art landscape around the key limitation — LM agarose in the expansion step. Note up front: no separate "Prior Art section" content was actually supplied in this prompt, so I'll build the analysis from (i) the references the patent itself cites, (ii) its own admissions about the state of the art, and (iii) live search results.

Let me verify two high-value prior-art threads — serial sectioning of agarose-embedded specimens (for the scan-and-slice claims) and any pre-2021 teaching of agarose in the expansion step itself.

Obviousness Analysis — U.S. Patent No. 11,333,588 B1 under 35 U.S.C. § 103

0. Scope caveat (please read first)

No separate "Prior Art section" content was supplied in this prompt. The materials I was given were (i) the patent's own text, (ii) a previously generated summary, and (iii) a litigation report. The Google Patents page does contain a "Prior art keywords / Prior art date" block, but that is metadata, not references — note especially that the listed "Prior art date: 2020-12-07" is simply the filing date copied over because the patent asserts no earlier priority (confirmed in the earlier summary). It is not a statement that a specific prior-art document exists.

Accordingly, I have built this § 103 analysis from three legitimate sources, and I flag which is which:

1. References cited inside the patent itself (Background/Definitions/Example 1): Chen et al. 2015; Tillberg et al. 2016 (proExM); Ku et al. 2016 (MAP); Gambarotto et al. 2019 (U-ExM); Chung et al. 2012/2013 (CLARITY); Susaki et al. 2014 / Tainaka et al. 2018 (CUBIC); Murakami et al. 2018 (CUBIC-X); Ertürk et al. 2012 (3DISCO); Reveles Jensen & Berg 2017; and the WO publications (WO 2015/127183; WO 2017/027367; WO 2017/027368; WO 2017/147435; WO 2017/190101; WO 2019/156957; WO/2019/241662; WO 2020/013833).
2. Applicant admissions in the specification and Table 1 — usable as prior art (MPEP § 2129; In re Fout).
3. Live search results (2026) for the specific narrow limitation.

Date discipline — this matters a lot here, and search indexes mix pre- and post-filing documents:

I did not find (within search limits) a pre-2021 reference that expressly puts low-melting agarose into the swelling/expansion bath itself. That gap is the whole ballgame, and I address it head-on in §6–§7.

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1. Person of ordinary skill in the art (POSITA)

A POSITA here is a person with an M.S. or Ph.D. in biomedical engineering, bioengineering, neuroscience, or a related field, or an M.D./Ph.D. neuroscientist, with 2+ years' hands-on experience in tissue clearing and/or expansion microscopy, or a B.S. with ~5 years of such experience — i.e., someone who has personally gel-embedded, cleared/digested, expanded and imaged tissue and who routinely reads Nature Methods / Nature Biotechnology / eLife protocols. Critically, this POSITA would know (a) the classic four-step ExM/CLARITY pipeline, (b) that expanded gels are ~99% water and mechanically fragile, and (c) that low-melting-point (LMP/LM) agarose is the standard lab matrix for immobilizing delicate specimens for cutting and imaging.

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2. The structural problem for the patentee: most of claim 1 is admitted prior art

Claim 1 has four steps. The specification's own Definitions section and Example 1 describe steps (a)–(c) as the conventional ExM/CLARITY workflow, e.g.:

"the processing steps typically involve sample embedding, which involves perfusing and cross-linking a specific type of polymer (such as acrylamide or sodium acrylate) throughout a fixed tissue, homogenizing the sample by protease digestion, adding water to expand the tissue and achieve tissue transparency…"

and

"Hydrogel Embedding — Fixed samples were pre-incubated in freshly prepared gelling solution (including acrylamide (AA), sodium acrylate (SA), N,N'-Methylenebisacrylamide (BA), NaCl in 1xPBS, and VA-044) for at least 24 hours at 4° C… Polymerization was then performed by incubating the tube at 37° C."

That is step (a) (pre-incubate in gelling solution with hydrogel precursor), step (b) (add VA-044 initiator; polymerize), and step (c) (SDS denaturation) described as the applicant's own starting materials and process. Under In re Fout / MPEP § 2129, an applicant's own specification statements about the prior art are treated as admitted prior art.

Consequence: the § 103 fight is almost entirely confined to step (d) — requiring the expansion solution to be "an aqueous solution of from 0.1% to 1% low melting point (LM) agarose."

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3. Element-by-element chart for claim 1

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4. The two strongest § 103 grounds

Ground 1 — MAP/U-ExM (steps a–c) + LM-agarose support of expanded gels + serial-sectioning art (step d)

Combination of:

• Ku et al. 2016, Nat. Biotechnol. (MAP) and/or Tillberg et al. 2016 (proExM) and/or Gambarotto et al. 2019 (U-ExM) → teach (a) high-monomer hydrogel embedding, (b) thermal polymerization, (c) SDS/heat denaturation as an alternative to protease digestion, and (d) swelling in water. The patent itself concedes MAP "preserves protein content by permeabilizing biological samples with high monomer concentrations … and by denaturing—rather than digesting—proteins prior to expansion."
• *Yu et al., eLife 46249 ("Expansion microscopy of C. elegans")* → in section Agar Immobilization (Optional): "If too much sample vibration or drifting is observed under a high-resolution objective (e.g. >= 40x) that affects image quality, immobilize expanded gel by adding droplets of lukewarm 2%(w/w) low-melt agarose solution…" (https://elifesciences.org/articles/46249). This teaches the exact function claimed: a low-melt agarose matrix added to an already-expanded hydrogel to stop drift/vibration during imaging.
• CLARITY protocols → teaching embedding cleared tissue in low-melting agar for trimming/sectioning (https://openspim.org/documents/3D_Tissue_Clearing_Handbook_3rd_Ed.pdf) and mounting clarified samples in 1% low melting point agarose for imaging (SAGE supplementary CLARITY protocol). This establishes both the material and the concentration overlap (1% is an endpoint of claim 1).
• ChroMS / Abdeladim 2019 (DOI 10.1038/s41467-019-09552-9) → microtome-based serial block-face imaging ("scan a layer → slice → image the adjacent layer") of agarose-embedded tissue, including discussion of agarose embedding and cross-linking to control slicing quality and lossless continuous imaging.
• LM agarose product literature (Lonza SeaPrep #50302, SeaPlaque #50104; Thermo UltraPure LMP #16500500) — the patent's own Definitions recite these and their properties (gelling ≤30 °C, melting ≤65 °C, high clarity).

Motivation to combine is documented in the art, not just imagined:

• The patent's own Background concedes expanded samples "become increasingly soft and fragile as they enlarge … can comprise as much as 99% water," that sample movement causes focus shift, that moving the stage slowly is "very time-consuming," and that "cutting can destroy, damage, or distort the sample."
• Locked-ExM states the same problem in print: "one of the common technical difficulties in implementing ExM is that its PA hydrogel dries and shrinks quickly when exposed to air, often leading to significant lateral drift during long imaging periods," and solves it by adding a second polymer network as mechanical support.
• Engerer et al., Nat. Biotechnol. 2016, already suggested the remedy: ExM samples "can become rather bulky… and re-slicing approaches (e.g., using an on-stage vibratome) are conceivable."

Ground 2 — proExM/ExM + CLARITY's LM-agarose cutting/mounting practice + routine optimization to 0.1–1%

Same as Ground 1 but substituting proExM as primary and CLARITY LM-agar embedment/mounting as the LM-agarose-teaching secondary. Motivation: CLARITY explicitly recommends "embedding the tissue in low melting agar and cutting the tissue down to the most practical form" — i.e., using the same class of matrix, for the same purpose (dimension control and cutting delicate hydrogel–tissue hybrids), in the same field.

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5. KSR rationales (why a POSITA would have combined)

Applying Graham v. John Deere / KSR Int'l v. Teleflex, at least five independent rationales support combination:

1. Known problem, known solution in the same field. The fragility/drift/uncuttable-ness of expanded gels was recognized in the ExM literature (see Ground 1 proof), and LM-agarose immobilization was already the field's go-to answer (C. elegans ExM protocol). KSR: "if a technique has been used to improve one device, and a person of ordinary skill in the art would recognize that it would improve similar devices in the same way, using the technique is obvious."
2. Use of a known material for its known property, with predictable results. LM agarose was known to (i) gel at ≤30 °C — i.e., remain liquid at the 37 °C used in the expansion step — and (ii) be highly transparent with a refractive index near that of water. Both properties are recited verbatim in the patent's own Definitions as the reasons for the choice. Where the reference discloses all the claimed structure and the difference is a substitution of a known material for its known qualities, the claim is obvious.
3. "Obvious to try" / finite number of identified, predictable solutions. Faced with the drift problem, a POSITA had a small, well-known menu of immobilization matrices (agarose, LMP agarose, gelatin, agar, collagen, Matrigel, hydrogels). LMP agarose was the most obvious because it remains liquid at handling temperature and is optically clear.
4. Routine optimization of a result-effective variable (concentration). The claimed range 0.1–1% encompasses the 1% LMP agarose concentration already used in CLARITY mounting, and the patent's own working examples use 0.35% and 0.5% with no data showing criticality at any boundary — and indeed the same patent mounts the sample in 2% LM agarose, outside its own claimed range. Absent evidence of a critical, unexpected limit, selecting a sub-range is routine optimization (In re Boesch; In re Peterson).
5. Design need / market pressure. The patent admits long "days of continuous scanning" were required for large samples and that existing approaches were "very time-consuming." That acknowledged pressure supplies the motivation.

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6. Dependent claims — a quick obviousness triage

Note on claim 12: the earlier-generated summary already flags that claim 12 reads "denaturing solution comprises an enzyme or a detergent," even though step (c) of claim 1 expressly requires homogenization by denaturation. The "enzyme" alternative is in tension with the base claim and with the specification, which treats protease digestion and heat/SDS denaturation as alternatives. This is a drafting inconsistency (like the previously reported "200 nM" vs. "200 mM" boric acid discrepancy) — worth raising both for claim construction and for any § 112(b) challenge.

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7. The patentee's best nonobviousness arguments (and the rebuttals)

A competent patentee has three arrows. They are not trivial, but none is decisive.

A. "The prior art mounts expanded gels in agarose; it never puts agarose in the swelling bath. That is a different step with a different result — the agarose is present as the gel expands, so it interpenetrates the network."

• Rebuttal 1 (claim language): step (d) requires only "incubating the denatured biological sample embedded in the hydrogel in an expansion solution … comprising an aqueous solution of from 0.1% to 1% LM agarose." Nothing in the claim text requires co-expansion or interpenetration. The claim is met by any expansion conducted in a dilute LM-agarose bath.
• Rebuttal 2 (order of addition): the difference is ordering/timing, and where the prior art teaches the same bath composition and the same specimen, reordering is obvious absent a new and unexpected result (In re Katz; MPEP § 2144.04). The patent's asserted benefit — the sample "wiggles less" — is the same benefit the C. elegans ExM protocol obtains by agarose addition.
• Rebuttal 3 (the 2% mounting example cuts both ways): the patent itself mounts the expanded sample in 2% LM agarose. If agarose's benefit were only available by co-swelling, the applicant's own mounting step would be superfluous.
• Concession: I found no reference that literally discloses LM agarose in the expansion solution, so there is no clean § 102 anticipation. This is a § 103 case, and the patentee's "different step" argument is the one that will carry the most weight with the PTAB.

B. Teaching away — agarose in contact with the sample causes optical problems.

• Evidence: post-expansion ExM protocols warn "Do not cover the sample itself with agarose as this will cause diffraction," and note agarose infiltration causes spherical aberration (see the 2022 STAR Protocols troubleshooting and the proExM protocol discussion at PMC12491153). But these are 2022 documents and so are not prior art; and the teaching is narrow (don't leave agarose between gel and coverslip), not a general discouragement.
• Worse for the patentee, the patent itself teaches the opposite — that LM agarose "has a high clarity, allowing expanded samples to attain a refractive index essentially equivalent to water," and it teaches rinsing the sample in water "to remove excess agarose" before imaging. So the patent's own position (clarity is fine; remove excess) neutralizes the teaching-away argument. A "teaching away" case requires the art to "criticize, discredit, or otherwise discourage" the claimed solution relative to alternatives (In re Fulton) — this art does neither.

C. Unexpected results / secondary considerations.

• The patent asserts, without data, that an LM-agarose-free hydrogel mounted in an agarose block "typically" deforms, and that imaging times drop (claims 26/82). There is no comparative experiment against an LM-agarose-free expansion bath, no quantified imaging-time comparison, and no evidence of commercial success, licensing, or industry praise in the record I reviewed. Under In re Geisler, attorney argument about advantages not supported by comparative data is entitled to little weight. This is the weakest of the three arrows.
• If, on the other hand, the applicant can show a synergistic/unexpected result (e.g., expansion factor and isotropy and reduced cutting distortion simultaneously, versus control), that could shift the calculus on claims 1–4. On the current record, no such data appears.

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8. Additional § 102/§ 103 lead that must be run down (flagged, not resolved)

The patent repeatedly cites WO 2020/013833 (also typed as "WO/2020/0013883" in the specification — a typo). It published in January 2020, before the Dec. 7, 2020 filing. Two urgent checks:

1. Does WO 2020/013833 disclose agarose (or any second polymer matrix) in the expansion/swelling step? If yes, it is likely a § 102(a)(1) or § 102(a)(2) reference and may anticipate claim 1 outright.
2. Common ownership / inventorship. WO 2020/013833 is cited as a foundational ExM application. If it shares common ownership or an inventor with the '588 patent, § 102(b)(2)(C)/§ 102(b)(1)(A) exceptions may remove it as prior art, or conversely, an earlier-effective-filing-date US national-phase publication could be a § 102(a)(2) reference. This determination is dispositive and I do not have the data to make it here — state it as an open item.

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9. Bottom line and confidence levels

• § 102 anticipation: No. I found no single reference disclosing all four steps, and in particular none putting LM agarose in the expansion solution. High confidence on "no anticipation found"; moderate confidence it does not exist (search-limited).
• § 103 on claim 1: Probably obvious, on the Ground 1 / Ground 2 combinations, with the strength resting entirely on (i) the patent's own admissions covering steps (a)–(c), (ii) the ExM and CLARITY literature's express recognition of the fragility/drift/cutting problem, and (iii) the field's established practice of low-melt-agarose immobilization, mounting and block-face sectioning of expanded or cleared hydrogel specimens. Moderate-to-high confidence, contingent on (a) verifying the publication dates of the eLife C. elegans protocol and the locked-ExM chapter, and (b) confirming whether WO 2020/013833 touches agarose in the swelling step.
• § 103 on claims 2–4, 7–11, 12 (detergent branch), 13–26: Obvious with high confidence — most are conventional conditions, known specimen identities, or steps the patent itself admits (4% LM agarose block; re-slicing).
• § 103 on claims 5–6 (dye co-added to the expansion bath): Moderate confidence. The dyes are standard; the step combination is supported by KSR, but the step-order/incompatibility argument available to the patentee is stronger here than for any other dependent claim. This is the sub-family most likely to survive if anything does.
• § 103 on claim 12's "enzyme" alternative: Not meaningfully assessable as drafted — the limitation is internally inconsistent with the "denaturing solution" requirement of claim 1.
• Claim-set caveat (carried forward from the earlier summary): the analysis above uses the claim wording rendered by Justia/Google Patents ("26 claims," single independent claim 1). Verify against the USPTO certified copy before relying on any of it for an invalidity contention or IPR petition.

Recommended next steps: (1) pull the certified claim set from Patent Center; (2) pull and date-check WO 2020/013833 and its US national-phase counterparts, and run the § 102(b)(2)(C) common-ownership analysis; (3) retrieve the C. elegans eLife protocol's exact e-pub date and add it as an express § 103 secondary reference with the "Agar Immobilization (Optional)" passage; (4) add ChroMS/Abdeladim (DOI 10.1038/s41467-019-09552-9) and Engerer 2016 as the scan-and-slice / re-slicing references for claims 10–11; (5) chase the underlying pre-2020 citations inside the 2025 Applied Physics Reviews "re-embedding expanded gels" passage to see whether a pre-2020 reference already teaches a second matrix interpenetrating an expanded gel.